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NEW BACTERIAL CELLULOSE NANOCOMPOSITES

PREPARED BY IN SITU RADICAL

POLYMERIZATION OF METHACRYLATE

MONOMERS

Tese apresentada à Universidade da Madeira com vista à

obtenção do grau de Mestre em Bioquímica Aplicada

Marisa Camacho Gonçalves Faria

Sob a orientação de:

Professora Doutora Nereida Maria Abano Cordeiro

Professora Doutora Carmen Sofia da Rocha Freire Barros

Faculdade de Ciências Exatas e da Engenharia

Universidade da Madeira

Funchal – Portugal

Departamento de Química e CICECO

Universidade de Aveiro

Aveiro, Portugal

2015

III

ACKNOWLEDGEMENTS

I would like to acknowledge every one that contribute in any way to the performance

of my master thesis, either at professional or/and motivational/personal level and made

possible their execution.

To my supervisor Prof. Dr. Nereida Cordeiro, for her constant support, guidance,

encouragement, patience and constructive critics which help me during my project and be

indispensable for the accomplishment of my project.

To my co-supervisor Prof. Dr. Carmen Freire for the support and all help in

recommending and understanding through my master thesis. To Dr. Armando Silvestre for

all the support and collaboration given in my project.

To University of Madeira for providing the facilities to carry out this project.

To Dr. Carla Vilela (University of Aveiro, Portugal), for performing the Infrared

Spectroscopy, NMR, X-ray diffraction, Scanning Electron Microscopy, Thermogravimetric

Analysis and Dynamic Mechanical Analysis and all support in data collection.

To Dr. Faranak Mohammadkazemi (Shahid Beheshti University, Iran) for running

the elemental chemical composition analyses.

To the laboratory technicians Paula Andrade and Paula Vieira, for availability and

support in the reagents and laboratory during my project.

To my colleagues and friends, Dina Maciel, Igor Fernandes, Micael Leça, Tomásia

Fernandes, Roberto Aguiar, Nuno Nunes, Emanuel Gouveia, Gabriel Freitas, Carla Alves,

Rogério Correia, José Rodrigues, Marisela Santos, Francisco Maciel, and Dr. Gabriel Leça

for all the motivation and help with their good dispositions and willingness through this

years.

To my family, Mom, Isabel, my sister, Elisabete, my brother, Roberto and João, and

my nephew, Afonso, for all support, encouragement and love all the time which help me to

continue and supporting me to go through.

Last but not the least, to my boyfriend, Ricardo Pascoal, for his endless support,

tireless patience and lovely sense of humor encouraging me all the time. Love you!

V

LIST OF PUBLICATIONS

Cordeiro, N., Ashori, A., Hamzeh, Y. & Faria, M. (2013) Effects of hot water pre-extraction

on surface properties of bagasse soda pulp. Materials Science and Engineering C, 33, 613–

617

Ashori, A., Cordeiro, N., Faria, M. & Hamzeh, Y. (2013) Effect of chitosan and cationic

starch on the surface chemistry properties of bagasse paper. International Journal of

Biological Macromolecules, 58, 343– 348

Cordeiro, N., Faria M., Abraham, E. & Pothan, L. A. (2013) Assessment of the changes in

the cellulosic surface of micro and nano banana fibres due to saponin treatment.

Carbohydrate Polymers 98, 1065– 1071

Cordeiro, N., Freitas, N., Faria, M. & Gouveia, M. (2013) Ipomoea batatas (L.) Lam.: A

Rich Source of Lipophilic Phytochemicals. J. Agric. Food Chem, 61, 12380−12384

Deepa, B., Abraham, E., Cordeiro, N., Mozetic, M., Mathew, A. P., Oksman, K., Faria, M.,

Thomas, S. & Pothan, L. A. (2015) Utilization of various lignocellulosic biomass for the

production of nanocellulose: a comparative study. Cellulose 22, 1075–1090

Castro, C., Cordeiro, N., Faria, M., Zuluaga, R., Putaux, J.L., Filpponen, I., Velez, L.,

Rojas, O.J. & Gañán, P. (2015) In situ glyoxalization during biosynthesis of bacterial

cellulose. Carbohydrate Polymers 126 (2015) 32–39

Mohammadkazemi, F., Faria, M., Cordeiro, N. In situ by«iosynthesis of bacterial

nanocellulose- CaCO3 hybrid composite: one.step process. 2015 (Submited)

VI

LECTURE

Moares, A.G.O., Faria, M., Silva, F.W., Cordeiro, N. & Amico, S.C. (2013) Efeito de

tratamentos químicos nas propriedades de superfície de fibras de carbono via cromatografia

gasosa inversa. 12° Congresso Brasileiro de Polímeros (12°CBPol) Brasil

VII

ABSTRACT

Bacterial cellulose/polymethacrylate nanocomposites have received attention in

numerous areas of study and in a variety of applications. The attractive properties of

methacrylate polymers and bacterial cellulose, BC, allow the synthesis of new

nanocomposites with distinct characteristics. In this study, BC/poly(glycidylmethacrylate)

(BC/PGMA) and BC/poly(ethyleneglycol)methacrylate (BC/PPEGMA) nanocomposites

were prepared through in situ free radical polymerization of GMA and PEGMA,

respectively. Ammonium persulphate (APS) was used as an initiator and N,N’-

methylenebisacrilamide (MBA) was used as a crosslinker in BC/PGMA. Chemical

composition, morphology, thermal stability, water absorption, mechanic and surface

properties were determined through specific characterization techniques. The optimal

polymerization was obtained at (1:2) for BC/PGMA, (1:2:0.2) ratio for BC/GMA/MBA

and (1:20) for BC/PPEGMA, with 0.5% of initiator at 60 ºC during 6 h. A maximum of 67%

and 87% of incorporation percentage was obtained, respectively, for the nanocomposites

BC/PGMA/MBA and BC/PPEGMA. BC/PGMA nanocomposites exhibited an increase

of roughness and compactation of the three-dimensional structure, an improvement in the

thermal and mechanical properties, and a decrease in their swelling ability and crystallinity.

On the other hand, BC/PPEGMA showed a decrease of stiffness of three-dimensional

structure, improvement in thermal and mechanical properties, an increase in their swelling

ability and a decrease the crystallinity. Both BC/polymethacrylate nanocomposites exhibited

a basic surface character. The acid treatment showed to be a suitable strategy to modifiy

BC/PGMA nanocomposites through epoxide ring-opening reaction mechanism.

Nanocomposites became more compact, smooth and with more water retention ability. A

decrease in the thermal and mechanical proprieties was observed.

The new nanocomposites acquired properties useful to biomedical applications

or/and removal of heavy metals due to the presence of functional groups.

Keywords: bacterial cellulose, nanocomposite, in situ radical polymerization,

glycidylmethacrylate, poly(ethyleneglycol)methacrylate, chemical treatment.

VII

RESUMO

Os nanocompósitos de celulose bacteriana/polimetacrilatos têm ganho interesse em

inúmeras áreas de estudo e para diversas aplicações. As propriedades atrativas dos

monómeros metacrilatos e da celulose bacteriana, BC, possibilitam aos novos

nanocompósitos adquirirem características interessantes para diversas aplicações. No

presente estudo, os nanocompósitos de BC/poli(glicidilmetacrilato) (BC/PGMA) e

BC/poli(etilenoglicol)metacrilato (BC/PPEGMA) foram obtidos através da polimerização

radical livre in situ do GMA e PEGMA, respetivamente, dentro da rede da BC usando o

persulfato de amónia (APS) como iniciador. O N,N’-metilenobisacrilamida (MBA) foi usado

como agente reticulante nos nanocompósitos de BC/PGMA. A composição química, a

estrutura morfológica, a estabilidade química, a absorção de água e as propriedades

mecânicas e de superfície foram determinadas. A polimerização ótima foi observada para

uma razão de (1:2) para a BC/PGMA, (1:2:0.2) para a BC/PGMA/MBA e a uma razão de

(1:20) para o BC/PPEGMA com 0.5 % de iniciador a 60 °C durante 6 h. A percentagem

máxima de incorporação de 67% e 87% foi obtida para os nanocompósitos

BC/PGMA/MBA e BC/PPEGMA, respetivamente. Os nanocompósitos de BC/PGMA

demonstraram uma estrutura rugosa e compacta, um melhoramento nas propriedades

mecânicas e térmicas, uma diminuição na sua capacidade de absorção de água e

cristalinidade. Por sua vez, os nanocompósitos de BC/PPEGMA apresentaram uma

diminuição na rigidez da rede, um melhoramento nas propriedades mecânicas e térmicas,

um aumento na sua capacidade de absorção de água e uma diminuição na cristalinidade. Os

nanocompósitos de BC/polimetacrilato exibiram um caracter básico à superfície depois da

incorporação dos metacrilatos. O tratamento químico ácido demonstrou ser uma estratégia

útil para a modificação dos nanocompósitos de BC/PGMA através do mecanismo de

abertura do anel epóxi. Os nanocompósitos tornaram-se morfologicamente mais compactos

e com a superfície mais lisa. No entanto, as propriedades mecânicas e térmicas decrescem e,

a superfície tornou-menos básica. Os novos nanocompósitos apresentam propriedades uteis

para aplicações biomédicas ou para a remoção de metais pesados.

Palavras-chave: celulose bacteriana, glicidilmetacrilato, poli(etilenoglicol)metacrilato,

nanocompósitos, polimerização radical livre in situ, tratamento químico.

XV

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .......................................................................................... III

LIST OF PUBLICATIONS ............................................................................................. V

ABSTRACT................................................................................................................. VII

RESUMO .................................................................................................................... VII

TABLES INDEX ........................................................................................................ XVI

LIST OF ABBREVIATIONS ................................................................................... XVIII

CHAPTER I. Overview of bacterial cellulose ................................................................... 2

1. Cellulose as Biopolymer ............................................................................................ 2

2. Bacterial Cellulose – Historical Outline ..................................................................... 4

2.1. Biochemistry of Bacterial Cellulose and Biosynthesis Pathways ........................... 4

2.2. Structure and Properties of Bacterial Cellulose .................................................... 6

2.3. Application of Bacterial Cellulose ....................................................................... 9

3. Bacterial Cellulose Nanocomposites ........................................................................ 10

3.1. BC nanocomposites through BC biosynthesis .................................................... 10

3.2. BC nanocomposites through in situ polymerization............................................ 12

3.3. Acrylate monomers used in BC nanocomposite ................................................. 14

4. Aim of the Study ..................................................................................................... 16

CHAPTER II. New bacterial cellulose/poly(glycidylmethacrylate) nanocomposites films by

in situ free radical polymerization ................................................................................... 20

1. Introduction ............................................................................................................ 21

2. Methodology .......................................................................................................... 22

2.1. Material ........................................................................................................... 22

2.2. BC nanocomposites preparation ....................................................................... 22

2.2.1. GMA and GMA/MBA polymerization ..................................................... 22

2.2.2. In situ free radical polymerization of GMA and GMA/MBA into BC ......... 23

2.3. BC nanocomposites characterization ................................................................ 24

2.3.1. Infrared Spectroscopy ................................................................................ 24

XVI

2.3.2. Energy-dispersive X-ray spectroscopy ......................................................... 24

2.3.3. Scanning electron microscopy .................................................................... 24

2.3.4. X-ray diffraction ........................................................................................ 24

2.3.5. 13Carbon Nuclear Magnetic Resonance....................................................... 25

2.3.6. Swelling ratio ............................................................................................. 25

2.3.7. Thermogravimetric analyses ....................................................................... 26

2.3.8. Dynamic mechanical analyses .................................................................... 26

3. Results and Discussion ............................................................................................ 26

3.1. In situ free radical polymerization reaction ........................................................ 26

3.2. Optimization of the in situ free radical polymerization ....................................... 28


3.3.1. Infrared and X-ray Spectroscopy ................................................................ 30

3.3.2. X-ray diffraction ........................................................................................ 32

3.3.3. 13Carbon Nuclear Magnetic Resonance....................................................... 33

3.3.4. Scanning electron microscopy .................................................................... 34

3.3.5. Swelling ratio ............................................................................................. 35

3.3.6. Thermogravimetric analyses ....................................................................... 36

3.3.7. Dynamic Mechanical properties ................................................................. 38

4. Conclusions ............................................................................................................ 39

CHAPTER III. Epoxide ring-opening of bacterial cellulose/poly(glycidylmethacrylate)

nanocomposites ............................................................................................................. 43

1. Introduction ............................................................................................................ 44

2. Methodology .......................................................................................................... 45


2.2. Chemical modification of BC nanocomposites .................................................. 45



3.1. Structural Analysis ........................................................................................... 48

3.2. Morphological Analyses ................................................................................... 49

3.3. Thermal and Mechanical Analyses ................................................................... 50

3.4. Surface properties ............................................................................................. 52

XVII

4. Conclusions ............................................................................................................ 56

CHAPTER IV. Novel bacterial cellulose/poly(ethyleneglycol) methacrylate nanocomposite

films obtained by in situ free radical polymerization ........................................................ 59

1. Introduction ............................................................................................................ 60

2. Methodology .......................................................................................................... 61

2.1. Material ........................................................................................................... 61




4. Conclusions ............................................................................................................ 68

CHAPTER V. Assessment of the surface properties changes of bacterial

cellulose/polymethacrylate nanocomposite films by Inverse Gas Chromatography ......... 70

1. Introduction ............................................................................................................ 71

1.1. Inverse gas chromatography ............................................................................. 71

1.1.1. Surface energy component ......................................................................... 72

1.1.2. Acid base character .................................................................................... 73

1.1.3. Isotherm measurements ............................................................................. 74

2. Experimental methodology ..................................................................................... 75

2.1. Material ........................................................................................................... 75

2.2. BC/PGMA and BC/PEGMA nanocomposites ................................................. 75

2.3. IGC measurements ........................................................................................... 76

2.4. Swelling ratio ................................................................................................... 77

2.5. Scanning electron microscopy ........................................................................... 77

2.6. Energy-dispersive X-ray spectroscopy ............................................................... 78


3.1. Dispersive component of surface energy ........................................................ 78

3.2. Heterogeneity ............................................................................................... 81

3.3. Surface area .................................................................................................. 83

3.4. Acid-base character ....................................................................................... 84

4. Conclusions ............................................................................................................ 87

XVIII

FINAL CONCLUSIONS .............................................................................................. 89

REFERENCES ............................................................................................................. 90

XIX

FIGURES INDEX

Figure 1. Molecular structure of cellulose where inter- (blue) and intra- (red) chain hydrogen

bonds dashed lines [reproduced from (14)]. ...................................................................... 3

Figure 2. Morphology characteristics of the different types of cellulose fibers [reproduced

from (15)]. ....................................................................................................................... 3

Figure 3. Schematic of the metabolic pathways of Glucanoacetobacter xylinum and the

assembly of cellulose molecules into nanofibrils [reproduced from (6)]. ............................. 6

Figure 4. A SEM of freeze-dried surface of bacterial cellulose gel [reproduced from (14)]. . 7

Figure 5. (A) X-ray patterns of BC films and (B) View along the direction 4 (i.e. [1ī0] for Iα

and [010] for Iβ) and the displacement of the hydrogen bonding sheets: blue Iα and red Iβ

[reproduced from (3, 13)]. ................................................................................................ 8

Figure 6. Different forms of BC produced by Gluconacetobacter sp. (A) Membrane, (B)

Irregular forms, (C) Sphere-like particles [reproduced from (36)]. ...................................... 8

Figure 7. Applications of bacterial cellulose: A – Tubes (blood vessel); B – Branched tube

fermented on a branched silicone tube (vascular grafts) [reproduced from (36)]; C –Dressing

(wound dressing) [reproduced from (7)]; D – Incorporation of AgNO3 nanoparticles

(antimicrobial activity); E –Paper (filter paper) and F – Colored with aniline blue dye

(bioadsorbent) [reproduced from (11)]. ............................................................................. 9

Figure 8. SEM images of surface morphology of (A) BC and (B) BC/Chitosan film in dry

form [reproduced from (41)]. ......................................................................................... 11

Figure 9. (A) Visual appearance of PVA: (a) without BC (b) BC/PVA nanocomposites and

(c) BC/PVA nanocomposites after chemical crosslinker and (B) Stress-strain behavior of

BC/PVA [reproduced from (39)].................................................................................... 12

Figure 10. (A) Visual appearance of BC/PAEM and all BC/PAEM/MBA nanocomposites.

(B) TGA thermograph of BC/PAEM nanocomposites. (C) Swelling of wet BC, BC/PAEM

and BC/PAEM/MBA nanocomposites [reproduced from (43)]. ..................................... 13

Figure 11. (A) Visual aspect of BC, BC/PHEMA and BC/PHEMA/PEGDA films and (B)

ADSCs proliferation in contact with BC and BC/PHEMA/PEDGA films [reproduced from

(40)]. ............................................................................................................................. 14

Figure 12. Molecular structure of glycidylmethacrylate (GMA). ..................................... 15

XX

Figure 13. Molecular structure of poly(ethylene glycol) methacrylate (PEGMA). ............ 16

Figure 14. Schematic reaction of in situ polymerization of GMA/MBA inside BC network.

..................................................................................................................................... 20

Figure 15. Scheme diagram of in situ free radical polymerization reaction of PGMA/MBA

inside of BC network. .................................................................................................... 27

Figure 16. Visual images of (a) BC, (b) BC/PGMA and (c) BC/PGMA/MBA

nanocomposites. ............................................................................................................ 28

Figure 17. Effect of reaction time (A), temperature (B), initiator (C), monomer (D) and

crosslinker (E) amount in BC in situ free radical polymerization. ..................................... 29

Figure 18. ATR-FTIR of native BC, BC/PGMA and BC/PGMA/MBA nanocomposites.

..................................................................................................................................... 30

Figure 19. XRD patterns of BC, BC/PGMA and BC/PGMA/MBA nanocomposites. ... 32

Figure 20. 13C NMR spectra of native BC, BC/PGMA and BC/PGMA/MBA

nanocomposites. ............................................................................................................ 34

Figure 21. SEM images of BC/PGMA and BC/PGMA/MBA nanocomposites. ............ 35

Figure 22. Swelling ratio in function of time of BC, BC/PGMA and BC/PGMA/MBA

nanocomposites. ............................................................................................................ 36

Figure 23. TGA and DTGA profile of BC/PGMA and BC/PGMA/MBA nanocomposites

and their controls. .......................................................................................................... 37

Figure 24. Storage modulus e tan versus temperature of BC and BC/PGMA

nanocomposites. ............................................................................................................ 38

Figure 25. Schematic diagram of the epoxide ring-opening mechanism of BC/PGMA

nanocomposites. ............................................................................................................ 47

Figure 26. 13C NMR spectra of BC/PGMA and BCP/PGMA/MBA nanocomposites before

and after chemical treatment .......................................................................................... 48

Figure 27. XRD patterns of BC/PGMA and BC/PGMA/MBA nanocomposites before and

after chemical treatment. ............................................................................................... 49

Figure 28. SEM images of BCGMA-CT and BCGMAMBA-CT nanocomposites

morphology in different perspectives. The (i), (ii), (iii) and (iv) images correspond to the

samples without chemical treatment. .............................................................................. 50

XXI

Figure 29. TGA and DTGA of BC/PGMA and BC/PGMA/MBA nanocomposites before

and after chemical treatment .......................................................................................... 51

Figura 30. Storage modulus e tan versus temperature of BCPGMA-CT nanocomposites.

..................................................................................................................................... 52

Figure 31. Heterogeneity profile with n-octane for BC/PGMA/MBA and BCPGMAMBA-

CT nanocomposites at 25 ºC. ......................................................................................... 54

Figure 32. Specific surface energy with polar probes: THF - tetrahydrofuran, DCM -

dichloromethane, ETOAc – ethyl acetate, ACN – acetonitrile of BC/PGMA/MBA and

BCPGMAMBA-CT nanocomposites at 25 ºC. ............................................................... 55

Figure 33. Schematic reaction of in situ free polymerization of PEGMA inside BC network.

..................................................................................................................................... 59

Figure 34. Schematic of in situ free radical polymerization reaction of PEGMA inside of BC

network [based on (86)]. ................................................................................................ 62

Figure 35. Effect of monomer (PEGMA) amount in BC in situ free radical polymerization.

..................................................................................................................................... 63

Figure 36. ATR–FTIR spectra of BC and BC/PPEGMA nanocomposite. ...................... 64

Figure 37. SEM images of BC and BC/PPEGMA nanocomposites morphology in different

perspectives. .................................................................................................................. 65

Figure 38. XRD patterns of BC and BC/PPEGMA nanocomposites. ............................. 66

Figure 39. Behavior of the swelling ratio in function of time of BC and BC/PPEGMA

nanocomposites. ............................................................................................................ 66

Figure 40. TGA and DTGA profile of BC and BC/PPEGMA nanocomposites. ............. 67

Figure 41. Storage modulus e tan versus temperature of BC/PPEGMA nanocomposites.

..................................................................................................................................... 68

Figure 42. Schematic representation of GC and IGC measurements (89). ....................... 72

Figure 43. SEM images of BC nanocomposites morphology (A) BC; (B) BC/PGMA; (C)

BC/PGMA/MBA and (D) BC/PPEGMA. ................................................................... 80

Figure 44. Schematic interactions of non-polar and polar probes with surface groups of

BC/PGMA/MBA nanocomposites. .............................................................................. 81

Figure 45. Heterogeneity profile with n-octane for native BC, (A) BC/PGMA and

BC/PGMA/MBA and (B) BC/PPEGMA nanocomposites at 25 ºC. ............................. 82

XXII

Figure 46. Specific surface energy of BC, BC/PGMA and BC/PGMA/MBA

nanocomposites with polar probes: THF - tetrahydrofuran, DCM - dichloromethane,

ETOAc – ethyl acetate and ACN – acetonitrile, at 25 ºC. ............................................... 85

Figure 47. Specific surface energy of BC and BC/PPEGMA nanocomposites with polar

probes: THF - tetrahydrofuran, DCM - dichloromethane, ETOAc – ethyl acetate, ACN –

acetonitrile, at 25 ºC. ..................................................................................................... 85

Figure 48. Heterogeneity profile with dichloromethane for (A) BC, BC/PGMA and

BC/PGMA/MBA and (B) BC and BC/PPEGMA nanocomposites, at 25 ºC. ................ 86

XVI

TABLES INDEX

Table 1. EDX analysis of elemental composition of BC, BC/PGMA and BC/PGMA/MBA

nanocomposites (wt %). ................................................................................................. 32

Table 2. Crystallinity index, 𝐼𝐶, and crystallite size, ACS, of BC/PGMA and

BC/PGMA/MBA nanocomposite. ................................................................................ 33

Table 3. Thermal degradation, 𝑇𝑑 (ºC), of BC/PGMA, BC/PGMA/MBA nanocomposites

and their controls in study. ............................................................................................. 38

Table 4. Thermal degradation, 𝑇𝑑 (ºC), of BC/PGMA and BC/PGMA/MBA

nanocomposites before and after chemical treatment. ..................................................... 51

Table 5. Surface properties of BC/PGMA/MBA and BCPGMAMBA-CT nanocomposites

at 25ºC. ......................................................................................................................... 53

Table 6. EDX analysis of elemental composition of BC and BC/PPEGMA nanocomposites

(wt %). .......................................................................................................................... 64

Table 7. Physical constants of all probes used in IGC experiments. ................................. 77

Table 8. Dispersive component of surface energy, 𝛾𝑠𝑑, swelling ratio, 𝑆𝑊, of BC, BC/PGMA

and BC/PPEGMA nanocomposites. ............................................................................. 79

Table 9. Surface area, 𝑆𝐵𝐸𝑇, monolayer capacity, 𝑛𝑚 and adsorption potential distribution

maximum, 𝐴𝑚𝑎𝑥., of BC, BC/PGMA, PC/PGMA/MBA and BC/PPEGMA

nanocomposites, at 25 ºC with n-octane. ........................................................................ 84

Table 10. Acid-base character, 𝐾𝐴and 𝐾𝐵 of BC, BC/PGMA BC/PGMA/MBA and

BC/PPEGMA nanocomposites, at 25 ºC. ...................................................................... 87

XVIII

LIST OF ABBREVIATIONS

∆𝑮𝒂𝒅𝒔 – Molar energy of adsorption

∆𝑮𝒔𝒑𝒔 – Specific component of the surface energy

∆𝑯𝒂𝒅𝒔𝒔𝒑

– Specific enthalpy of adsorption

∆𝑺𝒂𝒅𝒔𝒔𝒑

– Specific entropy of adsorption

𝑉𝑁 – Net retention volume

𝑨𝑵∗- Acceptor numbers

𝑨𝒎𝒂𝒙 – Adsorption potential

𝑲𝑨 – Acid constant

𝑲𝑩 – Basic constant

𝑺𝑩𝑬𝑻 – Surface area

𝑾𝑨 – Energy of adhesion

𝒂𝒎 – Cross area

𝒏𝒎 – Monolayer capacity

𝒑° - Saturation pressure

𝜸𝑳𝒅 – Dispersive component of the surface energy of the probe molecule

𝜸𝒔 – Surface energy

𝜸𝒔𝒅 – Dispersive component of the surface energy

ADSCs – Adipose-derived stem cells

AEM – 2-Aminoethyl methacrylate

APS – Ammonium persulphate

ATR – FTIR – Attenuated Total Reflection Fourier Transform Infrared Spectroscopy

ATRP – Atom transfer radical polymerization

BC – Bacterial cellulose

BC/PPEGMA – Bacterial cellulose/poly(ethyleneglycolmethacrylate)

BC/PGMA – Bacterial cellulose/poly(glycidylmethacrylate)

BC/PGMA/MBA – Bacterial cellulose/poly(glycidylmethacrylate) crosslinker with N,N’-

methylenebisacrilamide

BCPGMA-CT – Bacterial cellulose/poly(glycidylmethacrylate) after chemical treatment

XIX

BCPGMAMBA-CT – Bacterial cellulose/poly(glycidylmethacrylate) crosslinker with N,N’-

methylenebisacrilamide after chemical treatment

BuMA – Butyl methacrylate

c-di-GMP – Cyclic diguanylic acid

CPMAS 13C NMR – Solid-state Cross-Polarization Magic Angle Spinning Carbon-13

Nuclear Magnetic Resonance

DG – Degradation profile

DMA – Dynamic Mechanical Analysis

DMAEMA – N,N–dimethylaminoethylmethacrylate

Eʹ - Storage modulus

EDX – Energy-Dispersive X-ray Spectroscopy

EOEMA – 2-Ethoxyethyl methacrylate

FE–SEM – Field Emission Scanning Electron Microscopy

Glc – 1 – P – Glucose-1-phosphate

Glc – 6 – P – Glucose-6-phosphate

GMA – Glycidylmethacrylate

GMMA – Glycerol monomethacrylate

HEMA – 2-Hydroxyehtyl methacrylate

IGC – Inverse Gas Chromatograph

MBA - N,N’-Methylenebisacrilamide

MMA – Methyl methacrylate

PAAc – Poly(acrylic acid)

PDE-A and PDE-B – c-di-GMP phosphodiesterases A and B

PEG – Poly(ethylene glycol)

PEGDA – Poly(ethylene glycol)diacrylate

PEGMA – Poly(ethyleneglycol)methacrylate

PGMA – Poly(glycidylmethacrylate)

PVA – Poly(vinyl alcohol)

RAFT – Reversible addition-fragmentation chain transfer polymerization

SW – Swelling ratio

TGA – Thermogravimetric Analysis

XX

UDPG – Uracil diphosphate glucose

UGPase – UDPG-pyrophosphorylase

XRD – X-ray diffraction

𝑫𝑵 – Donor number

𝑵 – Avogadro’s number

𝑹 – Gas constant

𝑻 – Temperature

𝑻𝒅 – Thermal degradation

𝒂 – Area occupied by probe molecule

𝒄 – Heat of sorption

𝒏 – Amount adsorbed

𝒑 – Partial pressure

CHAPTER I

Bacterial cellulose and Nanocomposites world – General

Introduction

NEW BACTERIAL CELLULOSE NANOCOMPOSITES PREPARED BY IN SITU RADICAL POLYMERIZATION OF

METHACRYLATES MONOMERS

2

CHAPTER I. Overview of bacterial cellulose

1. Cellulose as Biopolymer

Originally from Greek and employed for the first time by the Swedish chemist

Berzelius in 1833, polymers (poli, many; meros, parts) are defined as large molecules

(macromolecules) synthesized from simple molecules, monomers, linked by covalent bonds

by polymerization processes (1-3).

Cellulose is the most abundant natural polymer because it is the main component of

the plant cell walls. It can be found in large amount in wood [above 50% (w/w)] and cotton

[above 94% (w/w)] (3). Due to its versatility and properties, cellulose can be applied into

different areas such as pulp and paper production, textiles, construction materials, cosmetics,

pharmaceutical industries as excipient and as a source for biofuel production (4, 5). Through

chemical processes like e.g. methylation and acetylation, cellulose derivatives can be

produced such as cellulose ethers and cellulose esters for other’s applications (3, 5-8). The

increase interest and demand for vegetable cellulose derivatives had augmented the

consumption of wood as a raw material, being the cellulose production approximately 1.5 x

1012 tons per year. This value represents a major fraction of the total biomass produced which

contributes to the deforestation and global environmental issues (4, 9, 10).

Cellulose was described for the first time by Anselm Payen in 1836 as “a resistant fibrous

solid that remains behind after treatment of various plant tissues with acids and ammonia, and after

subsequent extraction with water, alcohol, and ether” (11). It consists in a linear homopolymer

composed by D-glucose monomer glycosidically linked covalently in a β(1-4) conformation

through acetal functions, between the hydroxyl groups of C1 and C4 carbon atoms. It is

attributed a molecular formula of (C6H10O5)n where 𝑛 is the degree of polymerization of

glucose (Figure 1) (9, 11, 12). Two adjacent structural units constitute the basic repeating unit

called disaccharide cellobiose of the linear polymer (9). The linear configuration of cellulose

is stabilized by the intra-chain hydrogen bonds between the hydroxyl groups and the oxygens

of the adjacent ring molecules. Besides that, the interaction between hydroxyls groups and

oxygens of neighboring molecules via inter-chain hydrogen bonds promote the aggregation



3

of multiple cellulose chains resulting into nanofibrils. Combining both intra- and inter-chain

hydrogen bonding, turns the polymer relatively stable and give fibrils high axial stiffness (13).

Cellulose is insoluble in water, with high mechanical resistance and with a chemical

composition of 43.6–45 % carbon, 6.0–6.5 % hydrogen and the remainder oxygen. The length

of fibers depends of the source of cellulose and can range between 100 up to 10,000 nm (4).

Figure 1. Molecular structure of cellulose where inter- (blue) and intra- (red) chain hydrogen bonds dashed lines

[reproduced from (14)].

Cellulose with different morphologies can be obtained from conventional plant fibers

following adequate procedures as pulping processes (mechanical and chemical), acid

hydrolysis, steam explosion, high-intensity ultrasonification, among others (Figure 2) (4).

Figure 2. Morphology characteristics of the different types of cellulose fibers [reproduced from (15)].



4

2. Bacterial Cellulose – Historical Outline

Adrian J. Brown discovered in 1886 a gelatinous membrane as a product of microbial

fermentation of acetic acid from the “vinegar plant “or “mother” (6, 16, 17). Afterwards,

their chemical structure resembles that of native cellulose (cell-wall) through X-ray

diffraction. It was verified a crystallography typical of cellulose I, with two cellobiose units

arranged parallel in units cell and tended to have a specific planer orientation in dried film

(14). This gelatinous membrane was denominated as bacterial cellulose, BC, and the “vinegar

plant”, as Acetobacter (nowadays Gluconacetobacter) xylinum. As an exopolysaccharide, BC was

prompted a number of studies in order to better understand its biosynthesis pathways and

properties (18-20). BC can be produced in laboratory by several genera of bacteria such as

Gluconacetobacter, Entrobacter, Rhizobium, Agrobacterium and Sarcina sp during their

biosynthesis (12). Innumerable bacteria strains have been reported as BC producers. Known

as a gram-negative, aerobic, straight, slightly bent rods or ellipsoidal with dimensions of 0.6

x 4 µm, Gluconacetobacter strains are characterized as BC producers (6). For example,

Gluconacetobacter sacchari was reported as a high efficient producer of BC for the first time by

Trovatti et al (2011) with production yields of 2.7 g/L after 96h (21).

In recent years, BC has gained interest in numerous areas of study and in a variety of

applications due to its unique and specific properties such as high degree of crystallinity (80-

90%), high water retention capacity (99% of own weight), high purity (free hemicellulose and

lignin), high degree of polymerization (above 8000 wherein sometimes getting even 16,000

or 20,000), ultrafine fibrous network, high tensile strength, water holding ability, porosity,

biocompatibility, hydrophilicity, non-toxicity and a relatively simple production and efficient

in terms of costs (9, 11, 17, 22, 23). The BC display a typical Young´s modulus in the range

of 15-35 GPa with a tensile strength ranging between 200-300 MPa (9, 11).

2.1. Biochemistry of Bacterial Cellulose and Biosynthesis Pathways

Extensive research and reviews regarding the metabolic pathways of BC, genetic and

strains of cellulose-producing of BC have been reported (3, 24-27). Gluconacetobacter xylinum

is the most commonly model bacteria strain studied due to its ability to produce cellulose



5

from a wide range of carbon/nitrogen sources, being the rate of cellulose production

proportional to that of cell growth (6, 18, 28-30). Several studies indicate that the carbon

sources present in the medium influence the yield of BC as well as the bacteria strains and

the culture conditions: static and shaking (11, 29-35).

Two metabolic pathways operate in G. xylinum for BC production: the pentose

phosphate cycle (oxidation of carbohydrates) and the citrate acid cycle (oxidation of organic

acid and related compounds). G. xylinum is unable to metabolize glucose in the absence of

oxygen due to the lack or weakly present phosphofrutose kinase, which is required for

glycolysis. Glucose is the most common monosaccharides used as a carbon source in order

to obtain cellulose. Four fundamental enzymatic steps occur (i) phosphorylation of glucose

by glucokinase, (ii) isomerization of glucose-6-phosphate (Glc-6-P) to glucose-1-phosphate

(Glc-1-P) by phosphoglucomutase, (iii) synthesis of UDP-glucose (UDPG) by UDPG-

pyrophosphorylase (UGPase) and (iv) cellulose synthase reaction (Figure 3) (6, 18, 24).

UDPG is the immediate sugar nucleotide precursor of cellulose synthesis and for β(1-

4) glucan polymerization reaction. This enzyme allows an improved activity in BC producers

of c.a. 100x orders of magnitude (24). When disaccharides (sucrose or maltose) are used as

carbon source, the biosynthesis of BC initiates with the hydrolysis of disaccharides into

monosaccharides (fructose and glucose). Then, they are metabolized by the bacteria strain.

The molecular mechanisms of glucose polymerization into long and unbranched cellulose

chains are still nuclear, since the pathways of UDPG are relatively not well known. Cyclic

diguanylic acid (c-di-GMP) acts like an allosteric activator of the membrane-bound cellulose

synthase and display an important role in the biosynthesis of BC as a regulatory element (6).

The activity of c-di-GMP phosphodiesterases A and B (PDE-A and PDE-B) stopped the

activity of cellulose synthase. PDE-A cleaves the c-di-GMP to form pGpG which is

degraded, producing two molecules of 5’-GMP. The activity of PDE-A is selectively inhibited

by Ca2+ ions (24).



6

Figure 3. Schematic of the metabolic pathways of Glucanoacetobacter xylinum and the assembly of cellulose molecules into

nanofibrils [reproduced from (6)].

Cellulose is synthesized in microorganisms in two intermediary steps: (i) the

formation of β(1-4) glucan chains and (ii) the assembly and crystallization of cellulose chains.

The cellulose molecules are initially synthesized inside the bacteria and then spun through

cellulose export components to form protofibrils with a diameter c.a. of 2 – 4 nm. A ribbon

shaped microfibril of approximately 80 nm is assembled from these protofibrils. Cellulose

synthase is the catalyst for biosynthesis of cellulose, which polymerizes the glucose units into

the β(1-4) glucan chains (6).

2.2. Structure and Properties of Bacterial Cellulose

Structurally, BC possess an identical molecular formula (C6H10O5)n of the vegetable

cellulose as shown in Figure 1 (9, 11, 12). The process of biosynthesis of BC comprises two

main stages: linear polymerization of the glucose units catalyzed by the enzyme cellulose

synthase, followed by crystallization of the linear chains. The polymerization of β(1-4)

glucan starts from 300 to 10,000 glucose unit in order to form a linear polymer. This linear

polymer chain assembles with other adjacent chains to form aggregates (subfibrils) with 1.5



7

nm width, that further combine with another one to form semi-crystalline nanofibrils with

3.5 nm width. The nanofibrils combine between them, originating bundles and then the

macroscopic ribbon shapes (Figure 4) (9, 11). The size of nanofibrils and their spatial

arrangement strongly influences the BC crystallinity, which depends on two factors: source

(organism) and synthesis conditions (9, 11).

Figure 4. A SEM of freeze-dried surface of bacterial cellulose gel [reproduced from (14)].

Cellulose type I is the most common form biosynthesized and detected by X-rays

(Figure 5), consisting in two β(1-4) glucan chains oriented parallel to each other in a

monocyclic unit cell and arranged uniaxially (11). Native cellulose exhibited two different

structural crystals, cellulose Iα and Iβ. The cellulose Iα is crystallized in larger-size nanofibrils

while cellulose Iβ is formed in smaller-size nanofibrils being thermodynamically more stable

(3). Also, the allomorph forms differ in the unit cell: Iα show a triclinic unit and Iβ a

monoclinic unit cell. The ratio 𝐼𝛼 𝐼𝛽⁄ depends from species to species (3). The aggregates form

nanofibrils of width approximately 100 nm denominated fibrillary bands and these form the

tridimensional network structure. The 3-D micro and nanofibrillar structure of bacterial

cellulose influence the majority of its properties (9, 11). Bacterial cellulose, compared with

vegetal cellulose, had higher degree of polymerization, a higher degree of crystallinity and

higher size of crystallites (9, 11). These structural differences induce considerable differences

in terms of physical properties, particularly in terms of the mechanical strength (9, 11).



8

Figure 5. (A) X-ray patterns of BC films and (B) View along the direction 4 (i.e. [1ī0] for Iα and [010] for Iβ) and the

displacement of the hydrogen bonding sheets: blue Iα and red Iβ [reproduced from (3, 13)].

BC can be obtained in static and agitated culture media showing three different forms:

membrane, irregular shapes and sphere-like particles (Figure 6). BC membranes are produced

in static conditions at the air-liquid medium interface and have been commonly used for

different applications due to their suitable properties.

In contrast, irregular shapes and spheres of BC are produced under agitated culture.

Typically, BC produced in agitated culture media showed lower cellulose Iα content, lower

Young’s modulus, higher swelling and have potential to be used in food, healthcare and

materials applications (36).

Figure 6. Different forms of BC produced by Gluconacetobacter sp. (A) Membrane, (B) Irregular forms, (C) Sphere-like

particles [reproduced from (36)].

(A) (B)



9

2.3. Application of Bacterial Cellulose

BC is one of the finest examples of Nature’s art, with a singular morphology and

unique properties, and is gaining increasing interest as an excellent biopolymeric material to

be employed in a wide range of applications in different areas (Figure 7), namely in the:

(i) Biomedical and biotechnology fields: artificial skin, artificial blood vessels, artificial

cornea, heart valve prosthesis, artificial urethra, artificial bone, artificial cartilage,

artificial porcine knee menisci and delivery drug, hormone and protein,

reinforcement material for wound dressings, scaffolds for tissue engineering and soft

tissue replacement (7, 11);

(ii) Environmental and agricultural fields: dye decolorization, biadsorbent for heavy

metal removal and improvement of quality of soil with application of BC (11);

(iii) Electronic field: reinforcement transparent flat-panel (11);

(iv) Food fields: nata-de-coco manufacture (11);

(v) Industrial fields: papermaking (11);

(vi) Reinforcement as composites (11).

Figure 7. Applications of bacterial cellulose: A – Tubes (blood vessel); B – Branched tube fermented on a branched

silicone tube (vascular grafts) [reproduced from (36)]; C –Dressing (wound dressing) [reproduced from (7)]; D –

Incorporation of AgNO3 nanoparticles (antimicrobial activity); E –Paper (filter paper) and F – Colored with aniline blue

dye (bioadsorbent) [reproduced from (11)].



10

3. Bacterial Cellulose Nanocomposites

Nanocomposites were defined for the first time as “a multi-phase compound in which one

of the phases has a length scale in the nanometer range” by Roy and co-workers in 1980’s. A more

practical definition was introduced by Komarneni in 1992 that defined nanocomposites as

“composites of more than a Gibbsian solid phase where at least one-dimension is in the nanometer range

and typically all solid phases are in the 1-20 nanometer range” (37).

BC nanocomposites have been studied over time due to the excellent properties of

these materials. BC can be manipulated in order to improve mechanical performance and

biocompatibility, and can be employed in many forms from nano to macro scales for various

applications. They can be applied into plant biomimicking, biomedical, electrically

conductive materials, catalysis, optical and luminescent materials, proton conductive,

separating materials, antimicrobial materials, thermos-responsive, among many another’s

(36).

Different approaches for the preparation of BC nanocomposites have been reported

in literature (38-41), including: (i) incorporation of desire components in culture medium

during BC biosynthesis; (ii) In situ polymerization of monomers inside of the BC network and

(iii) blending with other polymeric materials. The first’s two approaches are applied in the in

situ preparation of nanocomposites and are described below.

3.1. BC nanocomposites through BC biosynthesis

One approach for the BC nanocomposites obtainment is the introduction of polymers

and/or desirable compounds into the culture medium during BC biosynthesis. This way, the

nanocomposite will be produced at the same time as the BC fibrils, through assembly of the

desire compounds. In fact, many compounds/polymers have already been incorporated in

BC culture media in order to obtain BC nanocomposites. Compounds/polymers such as

chitosan (41), carbon fibers, aloe-vera (42), glyoxal (38) or poly (vinyl alcohol) (PVA) (39).

Phisalaphong et al (2008) reported a new BC/chitosan film obtained through the

supplement of low molecular weight chitosan into the culture medium. The obtained

BC/chitosan nanocomposite exhibited a denser homogeneous fibril structure with smaller



11

pore diameter and higher surface area (Figure 8). Structurally, the introduction of chitosan

as a supplement into the culture medium didn’t change BC properties such as water vapor

transmission rates, average crystallinity index and antimicrobial ability (41).

A new nanostructured film of BC/aloe vera was reported by Saibuatong et al (2010)

through incorporation of aloe vera into the culture medium during biosynthesis of BC. This

assembly resulted in the enhancement of the mechanical properties, water absorption

capacity, water vapor transmission rate and crystallinity index. The BC/aloe vera

nanocomposite displayed an excellent compatibility and physical properties which make it a

good material with a wide range of application in medical areas (42).

Figure 8. SEM images of surface morphology of (A) BC and (B) BC/Chitosan film in dry form [reproduced from (41)].

Castro el at (2014) reported the production of BC/PVA nanocomposite films by

adding PVA to the culture media followed by chemical crosslinking. The chemical

crosslinking, after the biosynthesis, avoid the loss of the PVA matrix during the purification

steps improving the functional properties of the nanocomposites, reflected by the good

interaction between the PVA matrix and the reinforcement BC phases (Figure 9) (39).



12

Figure 9. (A) Visual appearance of PVA: (a) without BC (b) BC/PVA nanocomposites and (c) BC/PVA nanocomposites

after chemical crosslinker and (B) Stress-strain behavior of BC/PVA [reproduced from (39)].

Besides that, these authors reported the glyoxalization of BC films, by introducing

glyoxal into the culture medium during the BC biosynthesis. Glyoxalizated BC films showed

an increase in their hydrophobicity improving the BC properties while keeping its

crystallinity (38).

3.2. BC nanocomposites through in situ polymerization

Another approach to prepare BC nanocomposites is the in situ polymerization inside

the BC network. This method involves the absorption of a solution with the desire monomer,

the initiator and crosslinker (if used in the polymerization) into the BC network, followed by

the polymerization reaction. This approach is quite interesting because the final properties of

the nanocomposites can be simply tailored by using different monomers or monomers

mixtures.

In situ polymerization had already been used for the preparation of BC

nanocomposites with various methacrylates monomers such as 2-hydroxyehtyl methacrylate

(HEMA) (22, 40), 2-aminoethyl methacrylate (AEM) (43), glycerol monomethacrylate

(GMMA) (22) and 2-ethoxyethyl methacrylate (EOEMA) (22).

(A) (B)



13

Methacrylate monomers are versatile to prepare a series of BC/methacrylate

nanocomposites with different properties, due to their chemical structure variability, great

availability and easy polymerization (22).

Figueiredo et al (2015) reported the preparation of novel BC nanocomposites through

in situ polymerization of 2– aminoethyl methacrylate (AEM). Variable amounts of AEM and

N,N’-methylenebis(acrylamide) (MBA) used as a crosslinker were impregnated into the BC

membrane before the polymerization step. The BC/PAEM and BC/PAEM/MBA

nanocomposites were very homogeneous suggesting a good distribution of PAEM inside the

BC network, and were more translucent than native BC [Figure 10 (A)]. In addition, these

nanocomposites showed improved thermal stability and mechanical properties [Figure 10

(B)], a decrease in crystallinity and an increase of swelling ability [Figure 10 (C)] (43).

Furthermore, BC/PAEM nanocomposites (without crosslinker) display antibacterial

activity when in contact with a bacterial suspension of a bioluminescent Escherichia coli

demonstrating to be a good nanomaterial with properties for potential application as

antimicrobial wound dressing.

Figure 10. (A) Visual appearance of BC/PAEM and all BC/PAEM/MBA nanocomposites. (B) TGA thermograph of

BC/PAEM nanocomposites. (C) Swelling of wet BC, BC/PAEM and BC/PAEM/MBA nanocomposites [reproduced

from (43)].



14

In a different study (40), 2-hydroxyethyl methacrylate (HEMA) was used to prepare

a series of bacterial cellulose/poly(2-hydroxyethyl methacrylate) nanocomposites films by in

situ radical polymerization using poly(ethylene glycol) diacrylate (PEGDA) as a crosslinker

(40). As in the case of BC/PAEM nanocomposites, the translucency and homogeneity of the

BC/PHEMA and BC/PHEMA/PEGDA nanocomposite increased with the amount of

monomer and crosslinker polymerized inside the BC network [Figure 11 (A)]. The

nanocomposites exhibited improved thermal properties, good swelling ability and a decrease

in the storage tensile modulus. Furthermore, the nanocomposites are non-cytotoxic

providing favorable cell environment for optimal adhesion and proliferation of adipose-

derived stem cells (ADSCs) [Figure 11 (B)]. These makes them promising material for several

biomedical applications as for the design of 3D matrices with the purpose of maintaining a

cellular niche for stem-mediated tissue regeneration (40).

Figure 11. (A) Visual aspect of BC, BC/PHEMA and BC/PHEMA/PEGDA films and (B) ADSCs proliferation in

contact with BC and BC/PHEMA/PEDGA films [reproduced from (40)].

3.3. Acrylate monomers used in BC nanocomposite

Acrylate monomers such as acrylic acid, 2-ethylhexyl acrylate, 2-hydroxyethyl

methacrylate (22), methyl methacrylate (MMA), butyl methacrylate (BuMA) (44), N,N–

dimethylaminoethyl methacrylate (DMAEMA) (45) are typically used for polymerization

reaction due their high reactivity.

(B) (A)



15

Glycidylmethacrylate

Among acrylate monomers, glycidylmethacrylate (GMA) is of particular interest

because it presents two polymerizable groups: the epoxide and the methacrylate (Figure 12).

The double bond (C=C) of the methacrylate groups enables it to be subjected to a

polymerization process through radical polymerization reaction. The functional groups of

GMA can react with BC hydroxyl groups (46). Depending on the selective group

polymerizable or copolymerizable, the polymer acquires different reactivity. Most

commonly, the polymerization reaction mechanism involves the methacrylate groups while

the epoxide groups remaining unreacted (47). Moreover, this monomer offers spacer groups,

helping in the flexible movement of the polymer chain, favoring e.g. the adsorption of

compounds (48, 49). The hydrolysable ester group has been used due to its relatively low

toxicity, polarity, hydrophobicity, good reactivity, excellent biocompatibility and lower price

comparatively to others methacrylate monomers, make it an attractive monomer for

modification (47, 50-53).

These specificities open the possibility for several applications in the polymer

chemistry and technology such as for purification of lysozyme from chicken white egg (49,

50, 54), removal of heavy metals and chromate anions from aqueous solutions (48, 55-58),

and in coatings, matrix resins and adhesives (47, 59).

Figure 12. Molecular structure of glycidylmethacrylate (GMA).

GMA has already been reported in grafting polymerization from cellulosic backbones

using various polymerization systems such as free initiators (54, 55), redox systems like H2O2-

thiocarbonate, H2O2-Cu complex, reversible addition-fragmentation chain transfer

polymerization (RAFT) (60), photoinitiation, UV radiation and atom transfer radical

polymerization (ATRP) (52, 59).



16

Poly(ethylene glycol)methacrylate

Poly(ethylene glycol) (PEG) exhibited unique advantageous properties to be exploited

in biomedical and biotechnological devices due their low-toxicity, absence of antigenicity

and immunogenicity and inherent ability to prevent protein adsorption (61). Poly(ethylene

glycol)methacrylate (PEGMA), a PEG derivative, is another attractive methacrylate

monomer due to its amphiphilic nature. This property comes from its water-soluble PEG

side chain with a pendant hydroxyl group and its hydrophobic methacrylate group (Figure

13) (62). Due to these properties, PEGMA is one of the most attractive monomers used to

prepare biomedical materials such as drug carrier (63), microspheres for transient vascular

embolization (62), protein adsorption and immobilization of polysaccharides (64). It is also,

used for heavy metals removal (65, 66).

Different approaches of polymerization of PEGMA have been reported in the

literature such as photopolymerization (63, 67) and ATRP (61).

Figure 13. Molecular structure of poly(ethylene glycol) methacrylate (PEGMA).

4. Aim of the Study

In view of all issues described above, the aim of the present study is to prepare novel

BC nanocomposites for potential biomedical application through in situ free radical

polymerization of glycidylmethacrylate (GMA) and poly(ethyleneglycol)methacrylate

(PEGMA) using N,N’-methylenebis(acrylamide) (MBA) as crosslinker.

All BC nanocomposites were characterized in terms of structure, morphology,

thermal stability, water absorption, mechanical and surface properties by Attenuated Total

Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), CPMAS 13C NMR, X-ray

diffraction (XRD), Energy-Dispersive X-ray Spectroscopy (EDX), Field Emission Scanning



17

Electron Microscopy (FE-SEM), Swelling (SW), Thermogravimetric Analysis (TGA),

Dynamic Mechanical Analysis (DMA) and Inverse Gas Chromatography.

The present manuscript is laid out in four different chapters, each one corresponding

to scientific papers to be submitted briefly.

CHAPTER II

New bacterial cellulose/poly(glycidylmethacrylate)

nanocomposites films by in situ free radical polymerization


METHACRYLATE MONOMERS

20

CHAPTER II. New bacterial cellulose/poly(glycidylmethacrylate)

nanocomposites films by in situ free radical polymerization

Abstract

Novel bacterial cellulose/poly(glycidylmethacrylate) nanocomposites were prepared

by in situ free radical polymerization of glycidylmethacrylate using N,N’-

methylenebisacrilamide as crosslinker. The obtained nanocomposites were characterized in

terms of chemical structure, morphology, thermal stability, water absorption and mechanical

properties. The optimal conditions achieved for the polymerization were a proportional ratio

of 1:2:0.2 (bacterial cellulose/monomer/crosslinker), 0.5% (in respect to monomer) of

initiator at 60 ºC during 6 h. An incorporation percentage of about 67% was obtained for

these experimental conditions. BC nanocomposites exhibited a stiff and compact three-

dimensional structure. It was also observed an improvement in thermal and mechanical

properties, a decrease in

their swelling ability and

crystallinity. The new

nanocomposites possess

epoxy groups in their

structure which could react

with others molecules,

making them suitable for

applications in removal of

heavy metal or/and

proteins.

Keywords: bacterial cellulose, glycidylmethacrylate, in situ radical polymerization,

nanocomposites, morphology, thermal and mechanical properties.

Figure 14. Schematic reaction of in situ polymerization of GMA/MBA inside BC

network.



21

1. Introduction

Bacterial cellulose (BC) is a pure form of cellulose produced by several bacteria

including Gluconacetobacter sacchari which are a gram-negative, rod shaped and strictly aerobic

bacteria (16, 17, 21-23). BC confers unique and specific properties such as high degree of

crystallinity (80-90%), high water retention capacity (99%), ultrafine fibrous network, high

tensile strength, biocompatibility, hydrophobicity and non-toxicity. These properties stand

out BC as a good polymer to study having a relatively simple production thus cost efficient

(17, 22, 23, 68).

However, BC possess some limitations that restrict its applications as lack of

antibacterial properties, optical transparency and stress bearing capability (10). To overcome

these limitations, BC nanocomposites have been prepared, consisting in a BC network and

reinforcement materials (6, 10, 11, 69). The reinforcement materials improve BC’s biological

and physiochemical properties (10). BC nanocomposites have been synthetized through in

situ biosynthesis (38, 42), blended with other polymeric materials (12) and by in situ radical

polymerization (40, 43).

In the present study, it is exploited the in situ radical polymerization of methacrylate

monomers into the BC network. Glycidylmethacrylate (GMA) is a methacrylate monomer

with two polymerizable groups: the epoxide and the methacrylate groups (double bonds)

which offers a wide range of industrial applications in the polymer chemistry and technology

namely for purification of lysozyme from chicken white egg (49, 50, 54), removal of heavy

metals and chromate anions from aqueous solutions (48, 55-58), in coatings, matrix resins

and adhesives (47, 59). Besides that, GMA show a relatively low toxicity, polarity,

hydrophobicity and low price which make it an attractive monomer (47, 50-53). GMA has

been reported in grafting polymerization on cellulosic backbones using various

polymerization systems such as free initiators (54, 55), redox systems like H2O2-

thiocarbonate, H2O2-Cu complex, reversible addition-fragmentation chain transfer

polymerization (60), photoinitiation, UV radiation and atom transfer radical polymerization

(52, 59).



22

This work report the development of BC nanocomposite through in situ radical

polymerization of GMA into BC network using N,N’-methylenebisacrilamide (MBA) as

crosslinker and ammonium persulphate (APS) as initiator. The optimal conditions of

polymerization were determined and the new BC nanocomposites membranes were

characterized in term of structural, morphological, water ability, thermal, and viscoelastic

properties.

2. Methodology

2.1. Material

Wet bacterial cellulose (BC) membranes produced by Gluconacetobacter sacchari (70)

using standard conditions were used. Glycidylmethacrylate (GMA, 97%, with 100 ppm of

monomethyl ether hydroquinone as inhibitor), N,N’-methylenebisacrilamide (MBA, ≥

99.5%) and ammonium persulphate (APS, 98%) were used as monomer, crosslinker and

initiator, respectively. All reagents were purchased from Sigma-Aldrich and used as received.

Distilled water was used as solvent in all steps of the procedure.

2.2. BC nanocomposites preparation

2.2.1. GMA and GMA/MBA polymerization

A solution of 500 mg of GMA (monomer) and APS (initiator) (0.5% w/w GMA) in

distilled water and N2 atmosphere was prepared. This reaction mixture was placed at 60 ºC

for 6 hours to prepare the PGMA/MBA polymer. PGMA/MBA polymer was prepared

using the same conditions used for PGMA, but with addition of 20% (𝑤𝑐𝑟𝑜𝑠𝑠𝑙𝑖𝑛𝑘𝑒𝑟 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ )

MBA crosslinker to the monomer and initiator solution.



23

The obtained PGMA/MBA polymer after the reaction time was washed several times

with water, while PGMA was washed with methanol, and both were dried at 40 ºC

overnight, and stored after that in a desiccator until their characterization.

2.2.2. In situ free radical polymerization of GMA and GMA/MBA into BC

The in situ free radical polymerization of GMA inside the BC network was adapted

from the procedure described by Figueiredo et al. (43). An aqueous reaction mixture

containing the monomer (𝑤 𝑤𝑑𝑟𝑦 𝐵𝐶⁄ ) in a ratio of 1:2, 0.5% initiator (𝑤 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ ) and 20%

crosslinker (𝑤𝑐𝑟𝑜𝑠𝑠𝑙𝑖𝑛𝑘𝑒𝑟 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ ) (when used in reaction) was prepared. Wet BC

membranes were weight and about 60% of their water content was drained. The drained

membranes and reaction mixture were purged in N2 for 30 min. After that the reactional

mixture was added to the BC membranes and left for 1 hour at room temperature to occur

the incorporation of the monomer (and of the initiator and crosslinker) inside the BC

network. Then, the polymerization reaction take place in an oil bath for 6 hours at 60 ºC.

The obtained nanocomposite membranes were washed with distilled water during 1 hour for

8 times, dried at 40 ºC and stored in a desiccator until their characterization.

The percentage of polymer incorporation in the BC network was determined by the

sample increasing weight after polymerization and was calculated according to Eq. (1):

% 𝐼𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑤𝑚𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝐵𝐶−𝑤 𝐵𝐶

𝑤 𝐵𝐶 × 100 (Equation 1)

where 𝑊 𝐵𝐶 is the weight of dry bacterial cellulose (g) and 𝑊𝑚𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝐵𝐶 is the weight

of dry bacterial cellulose after in situ free radical polymerization (g).



24

2.3. BC nanocomposites characterization

2.3.1. Infrared Spectroscopy

The infrared spectra were obtained using Attenuated Total Reflection Fourier

Transform Infrared Spectroscopy (ATR-FTIR) using a Perkin Elmer FTIR System Spectrum

BX spectrophotometer equipped with a single horizontal Golden Gate ATR cell after 32

scans in the 4000−500 cm−1 range with a resolution of 4 cm−1.

2.3.2. Energy-dispersive X-ray spectroscopy

The semi-quantitative elemental chemical compositions of the samples were

determined by energy-dispersive X-ray spectroscopy (EDX). Semi-quantitative analyses (wt.

%) were done for elements (carbon, oxygen and nitrogen) and the 𝐶 𝑂⁄ ratio was obtained.

EDX experiments were conducted at an accelerated voltage of 5 kV in a Hitachi SU 8090

equipment.

2.3.3. Scanning electron microscopy

Scanning electron micrographs of the surface samples were obtained by Scanning

Electron Microscopy (SEM), with a HR-FESEM SU-70 Hitachi equipment operating at 1.5

kV operating in the field emission mode. Samples were deposited on a steel plate and coated

with carbon before analysis.

2.3.4. X-ray diffraction

The X-ray diffraction (XRD) measurements were carried out with a Phillips X’pert

MPD diffractometer using Cu Kα radiation. The peaks were deconvoluted using Pearson VII

peak functions (Peakfit software) for crystallinity index, 𝐼𝐶, appearance crystal size (ACS)

(38):



25

𝐼𝐶 = 1 −𝐼𝑎𝑚

𝐼002 𝑥 100 % (Equation 2)

where 𝐼𝑎𝑚 is the maximum peak intensity at 2θ around 22º, representing the

crystalline region, and 𝐼002 is the minimum peak intensity at 2θ around 18º, representing the

amorphous region. The ACS was calculated using Scherrer´s formula:

𝐴𝐶𝑆 =(0.9𝜆)

𝐹𝑊𝐻𝑀 cos 𝜃 (Equation 3)

where FWHM is the width of the peak at half the maximum height.

2.3.5. 13Carbon Nuclear Magnetic Resonance

Solid-state Cross-Polarization Magic Angle Spinning 13Carbon Nuclear Magnetic

Resonance (CPMAS 13C NMR) spectra were recorded on a Bruker Avance III 400

spectrometer operating at a B0 field of 9.4 T using 9 kHz MAS with proton 90° pulse of 3

microseconds and a time between scans of 3 seconds. 13C CPMAS NMR spectra were

acquired using a contact time of 2000 (2000) microseconds. 13C chemical shifts were

referenced with respect to glycine (C=O at 176.03 ppm).

2.3.6. Swelling ratio

The swelling ratio (𝑆𝑊) of the membranes was determined by immersing the samples

in distilled water at room temperature with a minimum of three replicas. The weight increase

was periodically measured during 48 hours. For each measurement, the samples were taken

out of the water, their wet surfaces immediately wiped dry in filter paper, weighted, and then

re-immersed. The SW was calculated using the equation:

𝑆𝑊(%) =(𝑊𝑠−𝑊𝑑)

𝑤𝑑 × 100% (Equation 4)



26

where, 𝑊𝑆 is the samples weight after swelling and 𝑊𝑑 is the weight of dry sample

before swelling.

2.3.7. Thermogravimetric analyses

Thermogravimetric analysis (TGA) were carried out with a Shimadzu TGA 50

analyzer equipped with a platinum cell. Samples were heated at a constant rate of 10 °C/min,

from room temperature to 800 °C, under a nitrogen flow of 20 mL/min.

2.3.8. Dynamic mechanical analyses

Dynamic mechanical analyses were performed using tension as deformation mode

(single strain) on a Tritec 2000 DMA (Triton Technologies). For the temperature sweeps, a

ramp rate of 2 ºC/min was used and samples were heated from –100 to 200 ºC, at a frequency

of 1 and 10 Hz, with a displacement of 0.005 mm.

3. Results and Discussion

3.1. In situ free radical polymerization reaction

Novel BC nanocomposites were obtained through in situ free radical polymerization

of GMA into the BC network using MBA as crosslinker and APS as initiator. A schematic

illustration of in situ polymerization reaction of GMA inside the BC network is illustrated in

Figure 15. The in situ radical polymerization of GMA occurs through a homolytic cleavage

via C=C, initiated by free radical provoked by the dissociation of APS (71).

The crosslinking of PGMA with MBA allows a better incorporation of polymer inside

the BC network and consequently the retention of more polymer avoiding its removal

through washing (40, 72). This incorporation can change the morphology and properties of



27

the native bacterial cellulose as perceptible on the visual images of the obtained BC/PGMA

and BC/PGMA/MBA nanocomposites (Figure 16).

Figure 15. Scheme diagram of in situ free radical polymerization reaction of PGMA/MBA inside of BC network.

Both synthetized BC/PGMA nanocomposites display an opaque white appearance

while native BC exhibits a milky white appearance after air drying. BC/PGMA exhibited as

malleable material while the BC/PGMA/MBA materials showed an increased stiffness,



28

being difficult to remove from the Erlenmeyer after polymerization, which predict new

thermal, morphologic and surface properties.

Figure 16. Visual images of (a) BC, (b) BC/PGMA and (c) BC/PGMA/MBA nanocomposites.

3.2. Optimization of the in situ free radical polymerization

The effects of the reaction time and temperature, and monomer, initiator and

crosslinker amount on the incorporation yield of the methacrylate polymer were studied in

order to establish the optimal conditions for the in situ free radical polymerization of GMA

and GMA/MBA inside of the BC network. The goal of this study was to obtain the

maximum incorporation of polymer and crosslinker inside BC tridimensional network.

The PGMA and PGMA/MBA incorporation percentage was calculated through

Equation 1. Figure 17 shows the results achieved for the different conditions tested. The in

situ free radical polymerizations were carried out for different reaction times, namely 60, 180

and 360 min. As shown in Figure 17 (A), the highest polymer incorporation was attained for

6 hours of reaction for BC/PGMA and BC/PGMA/MBA nanocomposites with 60.8% and

67.3% of polymer incorporation in BC network, respectively. The effect of temperature on

polymerization was evaluated at 50, 60 and 70 ºC and it was concluded that 60 ºC is the

optimum temperature in the range considered [Figure 17 (B)]. The diffusion of monomer,

initiator and crosslinking from the solution to the BC membrane influences the extension of



29

the polymerization reaction (56). Thus, it’s probable that the number of free radicals increase

with the temperature increase (56). Figures 17 (C), (D) and (E) show the effects of the amount

of initiator, BC/PGMA and BC/PGMA/MBA ratio, respectively. The best concentration

of initiator, BC/PGMA and BC/PGMA/MBA molar ratio (considering the amount of

polymer and crosslinker incorporated) tested were 0.5%, (1:2) and (1:2:0.2), respectively. The

initiator concentration is referred by other authors as a crucial factor for a successful of

polymerization due to the formation of free radicals in solution (73). A lower incorporation

percentage at lower concentration of initiator may be related with the deficit of the free

radicals and at higher concentration due to the self-combination of the free radicals, causing

a failure of chain initiator or enhanced rate of termination which balances the rate of

propagation (56).

Figure 17. Effect of reaction time (A), temperature (B), initiator (C), monomer (D) and crosslinker (E) amount in BC in

situ free radical polymerization.



30


Of all ratios of BC/PGMA and BC/PGMA/MBA nanocomposites obtained, the

ratio of (1:2) and (1:2:0.2) were chosen to be characterized as they were where the maximum

ratio polymerization was achieved.

3.3.1. Infrared and X-ray Spectroscopy

ATR–FTIR analysis was performed in order to confirm the occurrence of the

polymerization inside of the BC tridimensional network. Figure 18 shows the ATR–FTIR

spectra of BC, BC/PGMA and BC/PGMA/MBA nanocomposites.

Figure 18. ATR-FTIR of native BC, BC/PGMA and BC/PGMA/MBA nanocomposites.



31

The BC characteristic peaks are visible in all BC nanocomposites spectra at 3338,

2896, 1448, 1148, 1108 and 1060-1028 cm-1 corresponding to the free stretching vibration of

the (O–H) group, stretching vibration (C–H), in plan bending (O–H), stretching (C–O–C),

deformation (C–H), stretching (C–C) ring of cellulose and stretching (C–O), respectively (43).

The two peaks at 3296 cm-1 and 710 cm-1 correspond to the monoclinic Iα allomorph and the

peaks at 3240 cm-1 and 750 cm-1 were attributed to the triclinic Iβ form of BC (38). The

appearance of peaks at 1724, 844 and 754 cm-1, attributed to the vibrations of ester carbonyl

group (C=O) and epoxy group of the PGMA chains, respectively, confirms the incorporation

of PGMA in the BC network.

The absence of peak at 1660 cm-1 [stretching of methacrylate group (C=C)] indicates

that the in situ free radical polymerization occurred with success. The presence of the epoxy

groups of PGMA confirms the in situ polymerization of PGMA into BC only via C=C. The

addition of crosslinker, MBA, is visible by the peaks at 1650, 1450 and 1390 cm-1 that

correspond to (N–H) and (C–N) acrylamide units. Thus, the characteristic peaks of polymer

and crosslinker observed in the BC nanocomposites spectra evidences their incorporation

into BC network.

The elemental composition of the BC/PGMA and BC/PGMA/MBA

nanocomposites was determined by EDX analysis (Table 1). The major elements in all

samples were carbon (C) and oxygen (O). Based on the molecular formula, GMA and BC

have a 𝐶 𝑂⁄ ratio of 1.79 and 0.75, respectively, and MBA has a 𝐶 𝑂⁄ ratio of 3.02. It´s

expected that the C amount in BC/PGMA nanocomposite increase due to the PGMA

incorporation. According to the obtained results, an increase for 𝐶 𝑂⁄ ratio was observed in

the BC/PGMA nanocomposite. By the other hand, was observed an increase of N amount

in BC/PGMA/MBA nanocomposites with a 𝐶 𝑁⁄ ratio of 6.45. These data evidence the

success incorporation of PGMA and MBA into the BC network and are in agreement with

ATR-FTIR.



32

Table 1. EDX analysis of elemental composition of BC, BC/PGMA and BC/PGMA/MBA nanocomposites (wt %).

Samples 𝑪 𝑵 𝑶 𝑪 𝑶⁄ Ratio

BC 75.37 1.58* 23.05 3.30

BC/PGMA (1:2) 80.70 1.40* 17.90 4.50

BC/PGMA/MBA (1:2:0.2) 72.37 11.22 16.41 4.41

*impurities due to culture medium.

3.3.2. X-ray diffraction

Figure 19 presents the XRD profiles of BC, BC/PGMA and BC/PGMA/MBA

nanocomposites. Three peaks characteristic of BC were assigned in the diffraction angle of

15.4º, 17.1º and 22.9º corresponding to (1ī0), (110) and (200) crystallographic planes for

BC/PGMA and BC/PGMA/MBA. The decrease in the intensity indicates a decrease in the

crystallinity of BC with the introduction of PGMA and MBA. The peak were deconvoluted

in order to determine the crystalline dimensions and crystallinity index, 𝐼𝐶, showed in Table

2. As expected, the incorporation of the polymers into the BC network change the BC chain

organization and leads to a decrease in the crystallinity index and a decrease in the crystallite

size.

Figure 19. XRD patterns of BC, BC/PGMA and BC/PGMA/MBA nanocomposites.



33

Table 2. Crystallinity index, 𝐼𝐶, and crystallite size, ACS, of BC/PGMA and BC/PGMA/MBA nanocomposite.

Sample

ACS (nm)

𝑰𝑪 (%) (1ī0) (110) (200)

BC/PGMA 0.692 2.29 1.98 68.96

BC/PGMA/MBA 0.899 0.907 0.927 56.51

3.3.3. 13Carbon Nuclear Magnetic Resonance

Figure 20 shows the solid-state 13C NMR spectra of native BC, BC/PGMA and

BC/PGMA/MBA nanocomposites films. 13C NMR spectra of BC display the characteristic

resonances of cellulose at δ 65.2 (C-6), 71.4–74.3 (C-2,3,5), 90.0 (C-4) and 104.8 ppm (C-1).

The resonance at 15.74 ppm correspond to the α-methyl group and at 48.99 ppm to

the backbone methylene carbons atoms. The presence of these signals confirms the success

of polymerization inside BC. The methylenoxy group, methyne and methylene carbons of

the epoxy group exhibited signal at 65.49, 54.45 and 44.73 ppm, respectively. The resonance

signal at 176.99 ppm is attributed to the carbonyl ester carbons of PGMA. Similar results are

reported in the literature for PGMA (74-76).

The 13C NMR spectra of BC/PGMA nanocomposites evidence that the resonance

signals correspond of the sum of the starting components (BC and GMA). In

BC/PGMA/MBA, the absence of crosslinker resonance signal at 163.88 ppm (amide

carbonyl carbons) may be due to the lower content used in mixture reaction.

Furthermore, the absence of 13C reasonable signals typical of the monomer (GMA) at

127.75 ppm attributed to (C=C), confirming the success of the in situ polymerization of GMA

into the BC network previously indicated by ATR–FTIR spectra.



34

Figure 20. 13C NMR spectra of native BC, BC/PGMA and BC/PGMA/MBA nanocomposites.

3.3.4. Scanning electron microscopy

SEM analysis was carried out to study the effect of the incorporation of PGMA in the

morphology of BC. Figure 21 shows the SEM images of the surface and cross-section views

of BC nanocomposites. The surface morphology of BC/PGMA and BC/PGMA/MBA is

different when compared with that of native BC which confirms the change in the structural

BC network. The incorporation of the PGMA makes the BC network more compact and

smoother, although it’s still visible the entangled microfibrils characteristic of BC.

BC/PGMA/MBA surface exhibited a soft appearance with small spaces between particles

in a wide range of sizes (from sub-µm to several µm wide) and a microporous structure.



35

Figure 21. SEM images of BC/PGMA and BC/PGMA/MBA nanocomposites.

3.3.5. Swelling ratio

In order to evaluate the rehydration ability of the BC nanocomposites, swelling studies

were performed immersing the films in water during 48 h at room temperature. Figure 22

shows the swelling ratios of native BC and nanocomposites. The BC/PGMA and

BC/PGMA/MBA nanocomposites display a lower rehydration ability (32.8% and 31.3%,

respectively) when compared with native BC (137.7%) after 48 h of immersion. In these

study, the addition of a crosslinker promotes crosslinked points in PGMA chains and,

consequently, an increase in the number of crosslinking polymer network which results in a

decrease of swelling ratio due to the epoxy groups amounts of PGMA into the BC (77). The

hydrophobic behavior and interactions of BC/PGMA nanocomposites may promote a more

rigid network (78) which are supported and in agreement with SEM analyses and

macroscopic observation.



36

Figure 22. Swelling ratio in function of time of BC, BC/PGMA and BC/PGMA/MBA nanocomposites.

3.3.6. Thermogravimetric analyses

Thermogravimetric analyses were carried out with the aim to evaluate the thermal

stability and degradation profile of BC/PGMA and BC/PGMA/MBA nanocomposite

films. This knowledge is important in numerous applications where materials might be

submitted to high temperatures and applied to any material that exhibits a weight change

upon heating (79). Beyond the nanocomposites, the native BC and PGMA with and without

crosslinker controls were also analyzed for comparison. Figure 23 shows the TGA and

DTGA obtained for each samples (controls and nanocomposites) and Table 3 presents their

maximum degradation temperatures ( 𝑇𝑑).

Native BC membranes exhibit one main degradation step at about 347 ºC which is

typical of cellulose (40, 79). The minor mass loss at around 100 ºC is attributed to water

evaporation.



37

Figure 23. TGA and DTGA profile of BC/PGMA and BC/PGMA/MBA nanocomposites and their controls.

PGMA display a degradation profile with three main degradation steps at around 326,

388, 414 ºC and start their decomposition at 210 ºC. The first steps of degradation of PGMA

probably may be due to the: (i) depolymerization reaction and (ii) decomposition of esters

(72). The minor mass loss at around 414 ºC can be attributed to decomposition of units

involving side group scission of PGMA (71). The addition of crosslinker turn the polymer

more thermally stable and provokes a displacement of the degradation profile evidencing a

start decomposition at around 278 ºC and possessing a degradation profile at around 352,

398 and 408 ºC (Figure 23).

The TGA shows that incorporation of PGMA into the BC network influence the

thermal stability and degradation profile. The incorporation of PGMA and PGMA/MBA

into the BC increase their stability starting their decomposition, at around 275 ºC.

The increase of the 𝑇𝑑𝑖 in nanocomposites suggest a strong interaction and

compatibility between the PGMA and cellulose nanofibrils probably due to the establishment

of hydrogens bonds between BC nanofibrils and PGMA chains (40).



38

Table 3. Thermal degradation, 𝑇𝑑 (ºC), of BC/PGMA, BC/PGMA/MBA nanocomposites and their controls in study.

Samples 𝑻𝒅𝒊 𝑻𝒅𝒎𝒂𝒙𝟏 𝑻𝒅𝒎𝒂𝒙𝟐 𝑻𝒅𝒎𝒂𝒙𝟑

BC 266 347 --- ---

PGMA (1:0) 210 326 388 414

PGMA/MBA (1:0.2) 278 352 398 408

BC/PGMA (1:2) 273 321 363 413

BC/PGMA/MBA (1:2:0.2) 277 336 367 407

3.3.7. Dynamic Mechanical properties

The viscoelastic properties of BC/PGMA and BC/PGMA/MBA nanocomposites

were evaluated through dynamic mechanical analysis. Figure 24 shows the variation of the

storage modulus and tan 𝛿 of BC/PGMA and BC/PGMA/MBA nanocomposites.

Figure 24. Storage modulus e tan versus temperature of BC and BC/PGMA nanocomposites.

The storage modulus trace of the nanocomposites, E , shows only one transition. The

results obtained showed that the incorporation of PGMA provokes a decrease of storage



39

modulus when compared with native BC (40). This is due to the fact that PGMA is an

amorphous polymer, which decreases the crystallinity of the nanocomposites. The addition

of cross-linker leads to an increase of 11% in E ʹ and of 7 % in tan 𝛿 indicating that the cross-

linker make the nanocomposites with a tighter and higher stiffness BC network. The 𝑇𝑔

obtained from the tan 𝛿 peak also increase with the crosslinker addition from 90.2 to 96.5 ºC.

4. Conclusions

Bacterial cellulose/poly(glycidylmethacrylate) nanocomposites (BC/PGMA) were

obtained through in situ polymerization of glycidylmethacrylate using N,N’-

methylenebisacrilamide, MBA, as crosslinker and ammonium persulphate as initiator. The

maximum monomer incorporation into the bacterial cellulose occurs at (1:2) for BC/PGMA

and at (1:2:0.2) ratio for BC/PGMA/MBA, with 0.5 % of initiator, at 60 ºC during 6 h. The

incorporation of the PGMA makes the BC network more compact and smooth and allows

an improvement in their thermal and mechanical properties. It’s also observed that the

addition of a crosslinker allows a better retention of the polymer into BC network which

makes the new nanocomposite to exhibit a roughness and more compact three-dimensional

structure. Moreover, an improvement in thermal and mechanical properties was observed.

Both BC nanocomposites display a decrease in their water retention ability and in the

crystallinity degree. The new BC nanocomposites exhibited properties that can be applied for

absorbent materials for the removal of heavy metals and/or proteins due to the fact they

possess epoxy groups in their structure which could react with other molecules.

CHAPTER III

Epoxide ring-opening of bacterial

cellulose/poly(glycidylmethacrylate) nanocomposites



43

CHAPTER III. Epoxide ring-opening of bacterial

cellulose/poly(glycidylmethacrylate) nanocomposites

Abstract

New nanocomposites were prepared by epoxide ring-opening reaction mechanism of

bacterial cellulose/poly(glycidylmethacrylate) without (BC/PGMA) and with crosslinker,

N,N’-methylenebisacrilamide (BC/PGMA/MBA). To make it, the BC/PGMA and

BC/PGMA/MBA nanocomposites were subject to chemical treatment to promote epoxide

ring-opening reactions. A replacement of the epoxy groups for hydroxyls groups occurred in

an aqueous solutions at pH 3.5. After chemical treatment, the methacrylate nanocomposites,

BCPGMA-CT and BCPGMAMBA-CT, present a more compact and smoother structure. It

was also observed a decrease in thermal properties, mechanical properties, and crystallinity,

but an increase in their water retention ability. The surface became with less basic character

and occurred an increase in surface area. The reported methodology give rise to new and

interesting methacrylate nanocomposites.

Keywords: bacterial cellulose, glycidylmethacrylate, nanocomposites, chemical treatment,

epoxy-ring opening.



44

1. Introduction

Recently, bacterial cellulose (BC) has gained considerable attention as a source for the

development of new materials including nanocomposites due to its excellent properties such

as high purity, high degree of crystallinity (80-90%), high water retention capacity (99%),

ultrafine fibrous network and high tensile strength (17, 22). Combining the excellent

properties of BC with those of methacrylate monomers, such as high reactivity and

availability, bacterial cellulose/methacrylate nanocomposites have been prepared and

evaluated for different biomedical applications such as wound dressing (43).

Glycidylmethacrylate (GMA), among other methacrylate monomers, have received

particular attention because of the presence of two polymerizable groups: the epoxide and

the methacrylate groups. As polymer, it offers a wide range of industrial applications in

polymer chemistry and technology such as for purification of lysozyme from chicken white

egg (49, 50, 54), removal of heavy metals and chromate anions from aqueous solutions (48,

55-58), in coatings, matrix resins and adhesives (47, 59). Furthermore, GMA show a

relatively low toxicity, polarity, hydrophobicity and low price comparatively to other

methacrylate monomers which make them an attractive monomer for the polymer synthesis

(47, 50-53).

PGMA has been reported as grafted to different materials such as cellulose (49, 50)

via C=C, functionalizing the epoxy group, through epoxy ring-opening, with other molecules

as adsorbent material.

BC/PGMA nanocomposites, synthetized through in situ free radical polymerization

using N,N’-methylenebisacrilamide, MBA, exhibited a great retention of the polymer inside

the BC network. Without crosslinker, theses nanocomposites display a smoother and

compact network and with crosslinker, a rough, compact, dense and crosslinked network.

Both BC nanocomposites display an improvement in their thermal and mechanical

properties and, a decrease in the swelling ratio and crystallinity.

In the present study, a simple methodology was developed using the chemical

treatment that occurs in an aqueous solution at pH 3.5, to epoxide ring-opening of PGMA



45

inside BC network of BC/PGMA and BC/PGMA/MBA nanocomposites as a way to obtain

a microporous and crosslinked nanocomposite.

The obtained materials were characterized in terms of their structural and chemical

composition, crystallinity, morphology, thermal stability, water absorption, mechanical

properties and surface properties.

2. Methodology


Wet bacterial cellulose (BC) membranes produced by Gluconacetobacter sacchari (70)

using standard conditions were used. The BC/PGMA/MBA nanocomposites were obtained

through the methodology described in “Section 2.2. of Chapter II: New Bacterial

cellulose/poly(glycidylmethacrylate) nanocomposites films by in situ radical polymerization” of

present monography.

2.2. Chemical modification of BC nanocomposites

The chemical modification of BC/PGMA and BC/PGMA/MBA nanocomposites

was based in procedure described by Reis et al (2009) (80). Wet BC/PGMA and

BC/PGMA/MBA nanocomposites membranes were drained in order to remove the excess

of water and immersed in 2 mmol % HCl aqueous solution. Then, the reaction mixture was

placed at 140 ºC during 4 hours and under constant stirring (100 rpm). After this period, the

membranes were washed in distilled water until neutral pH, dried at 40 ºC and kept in a

desiccator until characterization.

Along this paper, the terminology of samples are described below: BC/PGMA–

bacterial cellulose membrane with poly(glycidylmethacrylate); BC/PGMA/MBA – bacterial

cellulose membrane poly(glycidylmethacrylate) crosslinking N,N’-methylenebisacrilamide;

BCPGMA-CT – chemical treatment of bacterial cellulose membrane with



46

glycidylmethacrylate and BCPGMAMBA-CT – chemical treatment of bacterial cellulose

membrane with glycidylmethacrylate crosslinking N,N’-methylenebisacrilamide.


The obtained BC/PGMA nanocomposite were characterized in terms of chemical

composition, morphology, thermal stability, water absorption, mechanic and surface

properties by ATR-FTIR, EDX, CPMAS 13C NMR, FE-SEM, XRD, TGA, SW, DMA and

IGC, respectively. The methodology applied for each methods is described in “Section 2.3.

of Chapter II: New Bacterial cellulose/poly(glycidylmethacrylate) nanocomposites films by in situ

radical polymerization” of present manuscript, expect the IGC analysis which is described

below.

Inverse Gas Chromatography measurements

The BC nanocomposites were packed in the standard glass silanized

(dymethyldichlorosilane; Repelcote BDH, UK) columns with 0.2 cm ID and 30 cm in length

by vertical tapping about 2 hours. The columns with the samples were conditioned 8 h at 40

ºC. After conditioning, pulse injections were carried out with a 0.25 mL gas loop. Four n-

alkanes (heptane, octane, nonane and decane) were used for measurements of the dispersive

component of the surface energy at 20, 25 and 30 ºC. Four polar probes (tetrahydrofuran,

dichloromethane, acetonitrile and ethyl acetate) were used to determine the specific

component of surface energy and acid-base character (𝐾𝐴 and 𝐾𝐵) at 25 ºC. The isotherm

experiments were undertaken with n-octane, tetrahydrofuran and dichloromethane at 25 ºC

for all samples tested. All experiments were carried out at 0% relative humidity with a helium

flow rate of 10 ml/min and at least in duplicate, producing an error less than 4%.

IGC measurements were carried out on a commercial inverse gas chromatograph

(iGC, Surface Measurements Systems, London, UK) equipped with flame ionization (FID)

and thermal conductivity (TCD) detectors. The iGC system is fully automatic with SMS iGC

Controller v1.8 control software. Data were analysed using iGC Standard v1.3 and



47

Advanced Analysis Software v1.25. The calculation methodology is described exhaustively

in Chapter V of the present manuscript.


BC/PGMA and BC/PGMA/MBA nanocomposites were subject to a chemical

treatment in order to promote the reaction of epoxy groups of the PGMA with hydroxyl

group of cellulose chain through epoxide ring-opening. The effect of attachment of PGMA

in structure, morphology, swelling, thermal, mechanical and surface properties was

evaluated through ATR-FTIR, 13C NMR, XRD, EDX, FE-SEM, SW, TGA, DMA and IGC

techniques.

A probably scheme illustrating the epoxide ring-opening mechanism of

BC/PGMA/MBA nanocomposites is shown in Figure 25.

Figure 25. Schematic diagram of the epoxide ring-opening mechanism of BC/PGMA nanocomposites.



48

3.1. Structural Analysis

After incorporation of PGMA with and without crosslinker into the bacterial cellulose

network, the chemical modifications occurred in aqueous solutions at pH 3.5 through

epoxide ring-opening mechanism. Reis et al (2009) reported a similar mechanism to explain

the chemical modifications of other macromolecules, poly(viny alcohol) (PVA) and

poly(acrylic acid) (PAAc)) with PGMA.

13C NMR spectra of BCPGMA-CT and BCPGMAMBA-CT nanocomposite films

was carried out and shown in Figure 26. The nanocomposites display characteristics

resonance signals of BC/PGMA nanocomposites with exception at 65.12 and 70.59 ppm

which is assigned to the carbon of ring-opening with an additional signal at 63.42 ppm for

BCPGMA-CT. These results suggest that the epoxide ring-opening forms, preferentially, a

hydroxyl group as reaction products. It’s also observed an increase in the intensity signal at

60.1 ppm which is attributed to the (–O–CH2–) atoms. The presence of these signals confirms

the epoxide ring-opening reaction by the binding of PGMA chain to hydroxyls groups of

cellulose backbone.

Figure 26. 13C NMR spectra of BC/PGMA and BCP/PGMA/MBA nanocomposites before and after chemical treatment



49

XRD analysis of the nanocomposites was carried out with the aim to know how the

chemical modifications affect the degree of crystallinity of nanocomposites. The crystallinity

index (𝐼𝐶) obtained for BCGMA-CT and BCPGMAMBA-CT was 38% and 43%,

respectively. The XRD patterns (Figure 27) and 𝐼𝐶 obtained for BCGMA-CT and

BCPGMAMBA-CT nanocomposites show that a significant decrease in the crystallinity

occurs after the chemical treatment, displaying a decrease in the intensity of peaks at 2θ (1ī0),

(110) and (200) when compared with BC/PGMA and BC/PGMA/MBA nanocomposites

(𝐼𝐶= 69% and 56%, respectively). This happens probably due to the attachment of PGMA

chain to cellulose backbone, which decrease the structural arrangement of BC fibrils.

Figure 27. XRD patterns of BC/PGMA and BC/PGMA/MBA nanocomposites before and after chemical treatment.

3.2. Morphological Analyses

SEM images reveal that the surface morphology changed with the chemical treatment.

The BC hydroxyls group reaction with epoxy ring of PGMA seems to make the BC network

surface more homogeneous probably due to the linkage between PGMA and cellulose chain

[Figure 28 (b) and (d)]. At surface, the microfibril of BC was more exposed to the surface in



50

both samples but more compact with chemical modification as shown in Figure 28 (a) and

(c). Thus, the chemical treatment of the BC /PGMA nanocomposites change their

morphology at surface and inside, turning it more compact and smooth which, consequently,

affect the properties of the BC nanocomposites.

Figure 28. SEM images of BCGMA-CT and BCGMAMBA-CT nanocomposites morphology in different perspectives.

The (i), (ii), (iii) and (iv) images correspond to the samples without chemical treatment.

3.3. Thermal and Mechanical Analyses

Figure 29 exhibit the TGA and DTGA achieved for nanocomposites and Table 4

show the maximum temperature of thermal degradation, 𝑇𝑑, obtained for each sample. The

minor mass loss at around 100 ºC is attributed to water evaporation of samples. BCPGMA-

CT nanocomposites display a degradation profile with three main degradation steps similar

to BC/PGMA nanocomposites. It´s observed the maximum degradation temperature of

PGMA which suggests that doesn´t occur the degradation of PGMA polymer provoked by

temperature or/and acid aqueous solution.



51

Figure 29. TGA and DTGA of BC/PGMA and BC/PGMA/MBA nanocomposites before and after chemical treatment

Table 4. Thermal degradation, 𝑇𝑑 (ºC), of BC/PGMA and BCP/PGMA/MBA nanocomposites before and after chemical treatment.

Samples 𝑻𝒅𝒊 𝑻𝒅𝒎𝒂𝒙𝟏 𝑻𝒅𝒎𝒂𝒙𝟐 𝑻𝒅𝒎𝒂𝒙𝟑

BC/PGMA 273 321 363 413

BC/PGMA/MBA 277 336 367 407

BCPGMA-CT 250 337 394 421

BCPGMAMBA-CT 265 332 398 417

Observing the TGA results, it’s evident that the epoxy ring of the BC/PGMA

nanocomposite affects the thermal stability of the fibers, and is related with the chemical

modification of BC network.

Dynamic mechanical analysis allows to evaluate the influence of chemical treatment

on the BC network viscoelastic properties. Figure 30 shows the variation of the storage tensile

modulus and tan 𝛿 of BCPGMA-CT and BCPGMAMBA-CT nanocomposites. It´s observed

that the storage modulus, E ʹ, started early the transition coinciding in the glass transition



52

zone in both nanocomposites. The chemical treatment increase the storage modulus

compared with BC/PGMA and BC/PGMA/MBA nanocomposites as well as the 𝑇𝑔

obtained from the tan 𝛿 peak (90.2 to 146.4 ºC and 96.5 to 99.2 ºC, respectively) (as described

in Section 3.3.6. of Chapter II). These results suggest that the reaction of epoxy group of PGMA

chain to cellulose backbone via (–OH), increase the amorphous content (supported by XRD

pattern). Moreover, these results also indicates that the nanocomposites became less rigid

than initial BC/PGMA nanocomposites.

BCPGMA-CT and BCPGMAMBA-CT exhibited distinct mechanical performance

which make these simple chemical treatment a good strategy to obtain nanocomposites for

new applications.

Figura 30. Storage modulus e tan versus temperature of BCPGMA-CT nanocomposites.

3.4. Surface properties

In order to study the effect of the chemical treatment in the nanocomposites surface,

IGC analysis was performed. This knowledge allows the prediction of its compatibility with

other materials. Parameters such as dispersive component of the surface energy, 𝛾𝑠𝑑, specific



53

component of the surface energy, ∆𝐺𝑠𝑠𝑝

, surface area, 𝑆𝐵𝐸𝑇, monolayer capacity, 𝑛𝑚,

adsorption potential, 𝐴𝑚𝑎𝑥., acid character, 𝐾𝐴 and basic character, 𝐾𝐵, were determined.

BCGMAMBA-CT exhibited a lower 𝛾𝑠𝑑 (37% less) when compared with

BC/PGMA/MBA (Table 5), suggesting that the surface became less hydrophobic due to a

decrease of the number or/and energy of the active sites.

Table 5. Surface properties of BC/PGMA/MBA and BCPGMAMBA-CT nanocomposites at 25ºC.

BC/GMA/MBA BCGMAMBA-CT

𝜸𝒔𝒅 (mJ/m2) 82.93 51.91

𝑲𝑨 0.13 0.10

𝑲𝑩 0.56 0.23

𝑲𝑩𝑲𝑨

⁄ 4.31 2.36

𝑺𝑩𝑬𝑻 (m2/g) 0.83 3.61

𝒏𝒎 (mmol/g) 2.18 9.52

Dispersive component of surface energy, 𝛾𝑠𝑑, acid, 𝐾𝐴, and basic, 𝐾𝐵, character, surface area, 𝑆𝐵𝐸𝑇, monolayer capacity, 𝑛𝑚.

The energetic profile of the surface heterogeneity is determined by the BET model (81)

through the injection of different concentrations of n-octane probe and provides an energetic

“map” of the nanomaterial surface. Figure 31 shows the heterogeneity profile for

BC/PGMA/MBA and BCPGMAMBA-CT nanocomposites at 25 ºC. After chemical

modifications, the nanocomposite shows a higher number of actives sites but less energetics

with a peak maximum at 9.71 kJ/Mol, suggesting that the chemical treatment decrease 𝛾𝑠𝑑

by the presence of less energetic actives sites at the surface of the nanocomposite.



54

Figure 31. Heterogeneity profile with n-octane for BC/PGMA/MBA and BCPGMAMBA-CT nanocomposites at 25 ºC.

The chemical treatment to the BCPGMAMBA-CT nanocomposite increase

significantly the 𝑆𝐵𝐸𝑇 and the monolayer capacity as shown in Table 5. This increase may be

due to the particular size decrease or the increase of surface porosity/rugosity (82). As seen

in SEM images, the surface became more roughness after the chemical treatment.

The specific component of surface energy, ∆𝐺𝑠𝑠𝑝

, was determined through injection of

polar probes and is present in Figure 32. The modification in the ∆𝐺𝑠𝑠𝑝

, indicates that the

chemical treatment change the polar groups in the nanocomposite. The nanocomposites

exhibited the maximum interaction with acetonitrile, which is an amphoteric probe, since it

reacts with both acid and basic groups in the nanocomposite surfaces. The chemical

treatment decrease the ∆𝐺𝑠𝑠𝑝

values for all probes which suggest that occurs a decrease in

polar active sites at the surface of the nanocomposite. This decrease was more significant for

the dichloromethane (acid probe: 43%) than for tetrahydrofuran (basic probe: 29%). These

results indicate that the BCPGMAMBA-CT nanocomposites exhibit more acid sites in the

surface due to the epoxide ring-opening which give rise to hydroxyls groups - the acid groups.



55

Figure 32. Specific surface energy with polar probes: THF - tetrahydrofuran, DCM - dichloromethane, ETOAc – ethyl

acetate, ACN – acetonitrile of BC/PGMA/MBA and BCPGMAMBA-CT nanocomposites at 25 ºC.

The ∆𝐺𝑠𝑠𝑝

is converted into acid-base number using the Guttmann’s concept (83),

and the results (Table 5) show that the chemical treatment decrease the base constant, 𝐾𝐵,

with a 𝐾𝐵 𝐾𝐴⁄ ratio 55% lower than BC/PGMA/MBA. Thus, the increase in the character

exhibited in BC nanocomposites derives from the epoxide ring-opening, which decrease the

basic groups (epoxide).

The know rehydration ability of the bacterial cellulose is attributed to the presence of

hydroxyls groups. The BCGMAMBA-CT nanocomposite exhibits a swelling ratio of 227%,

a considerable increase of c.a. 7 times plus when compared with BC/PGMA/MBA

nanocomposite (31%). These results are in agreement with 𝛾𝑠𝑑 trend data discussed previously

and reinforces the epoxide ring-opening process during the chemical treatment.



56

4. Conclusions

Natural polymers are able to react with many modifiers agents such as

glycidylmethacrylate, a bifunctional compound, in order to obtain new composites. Epoxide

ring-opening reaction was a suitable strategy to modify the bacterial

cellulose/poly(glycidylmethacrylate) nanocomposites. In order to do that, the BC/PGMA

and BC/PGMA/MBA nanocomposites were subjected to chemical treatment. The

morphology of BCPGMA-CT and BCPGMAMBA-CT nanocomposites is more compact

and smooth. A decrease in thermal and mechanical properties was observed while an

increase of the amorphous content and water retention ability in the new bacterial cellulose

nanocomposites was verified. The surface of BCPGMA-CT and BCPGMAMBA-CT

nanocomposites present a less basic character and an increase of the surface area was

observed. The reported methodology gives rise to new and interesting methacrylate

nanocomposites.

CHAPTER IV

Novel bacterial cellulose/poly(ethyleneglycol)methacrylate

nanocomposite films obtained by in situ free radical

polymerization



59

CHAPTER IV. Novel bacterial cellulose/poly(ethyleneglycol)

methacrylate nanocomposite films obtained by in situ free radical

polymerization

Abstract

New bacterial cellulose/methacrylate nanocomposite were prepared by the in situ free

radical polymerization of poly(ethyleneglycol)methacrylate, (PEGMA), inside the BC

network using ammonium persulphate, (APS), as radical initiator. Chemical composition,

morphology, thermal stability, water absorption and mechanic properties were determined.

The optimal conditions achieved for polymerization were a ratio of (1:20) for bacterial

cellulose/monomer with 0.5% of initiator during 6 h at 60 ºC. The maximum incorporation

percentage achieved was 87%. With the incorporation of the polymer, films became less stiff

and displayed a translucent-yellow color. The incorporation of the PPEGMA hydrophilic

chains originates an improvement of the thermal and mechanical properties and also their

swelling ability. A decrease in the crystallinity occurred due to the increase of the structural

entropy. The new nanocomposite acquires properties able to be applied in biomedical

materials.

Keywords: bacterial cellulose, PEGMA, in situ radical polymerization, nanocomposites

Figure 33. Schematic reaction of in situ free polymerization of PEGMA inside BC network.



60

1. Introduction

Recently, several studies have been reported using bacterial cellulose as a starting

component to obtain bacterial cellulose based nanocomposites. Bacterial cellulose produced

by Gluconacetobacter sp acquires excellent properties such as high purity, degree of crystallinity

(80-90%), high water retention capacity (99%), ultrafine fibrous network and high tensile

strength (17, 22). Besides that, they are biocompatible, hydrophilic and non-toxic (11).

Bacterial cellulose/methacrylate nanocomposites have been prepared with different

methacrylate monomers which provide a wide range of materials with adjustable properties

due to their variability in chemical structure, possibility of adapting the physicochemical

properties, great availability and easy to polymerization. (22) Wound dressing (43), design

of 3D matrices (40), removal of heavy metal and chromate anions from aqueous solutions

(48, 55-58), coatings, matrix resins and adhesives (47, 59) were some applications of bacterial

cellulose based methacrylate’s nanocomposites.

Poly (ethylene glycol) (PEG) display unique properties which are advantageous to

biomedical and biotechnological applications due its low-toxicity, absence of antigenicity

and immunogenicity and inherent ability to prevent protein adsorption (61).

Poly(ethyleneglycol)methacrylate (PEGMA), one PEG derivatives, is an attractive

methacrylate monomer due to its amphiphilic nature which comes from its water-soluble

PEG side chain with a pendant hydroxyl group and its hydrophobic methacrylate group (62).

PEGMA was used for biomedical materials such as drug carrier, microspheres for transient

vascular embolization (62), protein adsorption and immobilization of polysaccharides (64)

and heavy metal removal (65, 66).

PPEGMA can be produced by different polymerization approaches such as

suspension polymerization (65, 84), UV-initiated free radical polymerization (85) and atom

transfer radical polymerization (ATRP) (61).

The present study reports the development of BC nanocomposite through in situ

radical polymerization of PEGMA into BC network using APS as initiator. The optimal

polymerization conditions were determined and the obtained BC/PPEGMA nanocomposite

characterized in terms of chemical composition, morphology, thermal stability, water

absorption and mechanic properties.



61

2. Methodology

2.1. Material

Wet bacterial cellulose (BC) membranes using in standard conditions was used (70).

Poly(ethyleneglycol)methacrylate (PEGMA, 97%, with 100 ppm of monomethyl ether

hydroquinone as inhibitor, average of Mn = 500) and ammonium persulphate (APS, 98%)

were used as monomer and initiator, respectively. All reagents were purchased from Sigma-

Aldrich and used as received. Distilled water was used as a solvent in all procedure steps.


The in situ polymerization of PEGMA inside the BC network was adapted from the

procedure described by Figueiredo et al (43). Wet BC membranes were weight and 60% of its

water content was drained. A reaction mixture containing the monomer (𝑤 𝑤𝑑𝑟𝑦 𝐵𝐶⁄ ) in a

ratio of 1:20, 0.5% initiator (𝑤 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ ) was prepared in an aqueous solution and purged

in N2 for 30 min. as well as the drained membrane. After that, the reactional mixture was

added to the BC membrane and left for 1 hour at room temperature to occur the monomer

incorporation (and of the initiator and crosslinker) inside the BC network. Then, the

polymerization reaction was left reacting for 6 hours at 60ºC. The obtained nanocomposite

membranes were washed with distilled water during 1 hour for 8 times, dried at 40 ºC and

stored in a desiccator until their characterization.

The optimum percentage of polymer incorporation in the BC was determined by the

sample increasing weight after polymerization and was calculated according to Eq. (1)

% 𝐼𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑤𝑚𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝐵𝐶−𝑊

𝑊 (Equation 5)

where 𝑊 is the weight of dry bacterial cellulose (g) and 𝑊𝑚𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝐵𝐶 is the weight of

the dry bacterial cellulose after in situ radical polymerization (g).



62


The obtained BC/PPEGMA nanocomposite was characterized in terms of chemical

composition, morphology, thermal stability, water absorption and mechanic properties by

ATR-FTIR, XRD, EDX, FE-SEM, TGA, SW and DMA, respectively. The methodology

applied for each methods is described in “Section 2.3. of Chapter II: New Bacterial

cellulose/poly(glycidylmethacrylate) nanocomposites films by in situ radical polymerization” of

present monography.


BC/PPEGMA nanocomposites were prepared by the in situ free polymerization of

PEGMA incorporation into the BC network using APS as initiator. The schematic

representation of the polymerization is shown in Figure 34.

Figure 34. Schematic of in situ free radical polymerization reaction of PEGMA inside of BC network [based on

(86)].



63

The in situ radical polymerization of PEGMA occurs through a homolytic cleavage

via C=C, initiated by free radical provoked by the dissociation of APS (71).

Different reaction conditions were studied. The optimal conditions (considering the

amount of polymer incorporated) determined for this polymerization were a ratio of (1:20)

for bacterial cellulose/monomer with 0.5% of initiator at 60 ºC during 6 h resulting in a

polymer incorporation percentage of about 87% into the BC network (Figure 35).

Figure 35. Effect of monomer (PEGMA) amount in BC in situ free radical polymerization.

The obtained BC/PPEGMA nanocomposites display a translucent-yellow color

while native cellulose exhibits a milky white appearance. It’s also observed a decrease of

stiffness of the BC network, suggesting that the incorporation of polymer occurs into the

cellulose network.

This new nanocomposite was characterized in terms of structure, morphology,

rehydration ability, thermal and mechanical properties. ATR–FTIR analysis was performed

to confirm the incorporation of polymer inside of BC tridimensional network. Figure 36

shows the ATR–FTIR spectra of BC and BC/PPEGMA (1:20).



64

Figure 36. ATR–FTIR spectra of BC and BC/PPEGMA nanocomposite.

The ATR–FTIR spectra of the BC/PPEGMA nanocomposites exhibited, in addition

to the characteristic peaks of native BC (described in Section 3.3.1. of Chapter II) a absorption

band at 1720 cm-1, attributed to the carboxyl groups of methacrylate ester units (O–C=O).

Additionally, the intensification of the absorption at 2894 cm-1, assigned to the vibrations of

the CH, CH2 and CH3 and at 3340 cm-1, attributed to the PEG terminal hydroxyl groups (65,

85), confirm the success of incorporation of PPEGMA into the BC network. Moreover, the

absence of the characteristic C=C stretching vibrations at 1660 cm-1 indicates that the

polymerization of PEGMA occurs into the three-dimensional network.

A decrease of 12% in the 𝐶 𝑂⁄ ratio in BC/PPEGMA nanocomposites, determined

by EDX (Table 6) evidence the increase of hydroxyl groups (–OH) and ester groups (O–C=O)

into the network of BC, arising from the presence of PPEGMA in BC network.

Table 6. EDX analysis of elemental composition of BC and BC/PPEGMA nanocomposites (wt %).

Samples 𝑪 𝑶 𝑪 𝑶⁄ Ratio

BC 75.37 23.05 3.30

BC/PPEGMA (1:20) 73.14 25.23 2.90



65

As expected, the incorporation of hydrophilic groups from PPEGMA into the three-

dimensional BC network leads to changes in the morphology of the membranes as revealed

by SEM analysis (Figure 37). Comparatively with native BC, BC/PPEGMA

nanocomposites display a denser, compact and homogeneous surface morphology with

scattered between cellulose microfibrils. Inside of the network, thin laminated-type flakes

leaves were spread throughout the network. BC/PPEGMA nanocomposite network exhibit

particles with a wide range of sizes (from sub-µm to several µm wide). The SEM images

evidences that the incorporation of PPEGMA only occurs inside of the network.

Figure 37. SEM images of BC and BC/PPEGMA nanocomposites morphology in different perspectives.

The incorporation of PPEGMA can decrease the crystallinity of BC network due to

the BC fibrils disorganization. In the XRD profile of BC/PPEGMA, only one characteristic

diffraction peak of BC at 2𝜃 of 22.9º, corresponding to the (200) crystallographic planes was

observed (Figure 38). The crystallinity index obtained for BC/PPEGMA was 39.40% with

smaller crystallite dimensions (0.877 nm). This high decrease in the crystallinity index is in

line with the 87% of PEGMA incorporation.



66

Figure 38. XRD patterns of BC and BC/PPEGMA nanocomposites.

In order to evaluate the absorption of water and rehydration ability of the new BC

nanocomposites, the swelling studies were performed by immersion of the membranes in

water during 48 h at room temperature. Figure 39 shows the swelling ratios of native bacterial

cellulose and of the new BC/PPEGMA nanocomposites. Both samples absorbed water in

different ratios, reaching an equilibrium at 24 h. The introduction of PPEGMA chain with

hydroxyls groups (–OH) and ester groups (O–C=O) promotes an increase in the

hydrophilicity character of the samples, exhibiting an increment of 213% in the water

absorption when compared with native BC.

Figure 39. Behavior of the swelling ratio in function of time of BC and BC/PPEGMA nanocomposites.



67

The thermal stability and degradation profile of BC/PPEGMA nanocomposites was

evaluated by thermogravimetric analysis as shown in Figure 40. Native bacterial cellulose

membranes were also analyzed for comparison purposes. As described in Section 3.3.5. of

Chapter II, native BC exhibit one main degradation step with maximum degradation

temperature at about 347 ºC (42, 80). The addition of PPEGMA decrease the stability when

compared to BC, starting their decomposition at around 266 ºC and exhibited a degradation

profile with two main step degradation at around 342 and 402 ºC.

Figure 40. TGA and DTGA profile of BC and BC/PPEGMA nanocomposites.

The influence of PPEGMA incorporation on the viscoelastic properties of the novel

BC/PPEGMA nanocomposites was evaluated by DMA. Figure 41 shows the variation of

the storage tensile modulus and tan 𝛿 of BC/PPEGMA nanocomposites films. The storage

modulus, E ʹ, shows only one transition that start for a temperature lower than -43% (before

the beginning of the analysis). These results show that the incorporation of PPEGMA

originates a remarkable decrease in the E ʹ compared with native BC (40) due to fact that

PEGMA incorporation lead to a decrease of the film crystallinity as obseeved by XRD

analysis. These results indicated that the incorporation of PPEGMA into the BC network

results in a decrease of the rigidity of the membrane.



68

Figure 41. Storage modulus e tan versus temperature of BC/PPEGMA nanocomposites.

4. Conclusions

New bacterial cellulose/poly(ethyleneglycol)methacrylate nanocomposite,

BC/PPEGMA was obtained by in situ radical polymerization of PEGMA inside the BC

network. BC/PPEGMA nanocomposite display a translucent yellow appearance and have

new thermal, morphologic and surface characteristics. The incorporation of the hydrophilic

PEGMA inside the BC network lead to a decrease of the stiffness of three-dimensional

structure, improve the thermal and mechanical properties, increase their swelling ability as

well as of amorphous content.

CHAPTER V

Assessment of the surface properties changes of bacterial

cellulose/polymethacrylate nanocomposite films by Inverse Gas

Chromatography



70

CHAPTER V. Assessment of the surface properties changes of

bacterial cellulose/polymethacrylate nanocomposite films by

Inverse Gas Chromatography

Abstract

Bacterial cellulose/polymethacrylate nanocomposites have been receiving attention

in the last years due to the availability and attractive properties of methacrylate monomers.

In this work, bacterial cellulose/poly(glycidylmethacrylate) and bacterial

cellulose/poly(ethylene glycol)methacrylate nanocomposites were prepared by in situ free

radical polymerization. Inverse Gas Chromatography was used to study surface properties

namely surface energy, surface area, heterogeneity and acid-base character. The results show

the successful incorporation of methacrylate polymers into the bacterial cellulose. The

incorporation of glycidylmethacrylate decrease the hydrophilicity and increase the porosity

and roughness of the bacteria cellulose network. In the case of

poly(ethyleneglycol)methacrylate incorporation, an increase of hydrophilicity and a decrease

of the porosity and roughness, was observed. Both methacrylate polymers incorporation

made the bacterial cellulose film surface more basic and consequently more reactive with the

acid compounds, thus allowing new applications.

Keywords: Nanocomposites, bacterial cellulose, poly(glycidylmethacrylate),

poly(ethyleneglycol)methacrylate, surface properties, IGC.



71

1. Introduction

Nanocomposites are defined as ‘composites of more than a Gibbsian solid phase where at

least one-dimension is in the nanometer range and typically all solid phases are in the 1-20 nanometer

range’ by Komarneni in 1992 . Recently, several studies have been reported using bacterial

cellulose as a starting component and/or the base to obtain bacterial cellulose

nanocomposites. Bacterial cellulose possesses excellent properties such as high purity, degree

of crystallinity (80-90%), high water retention capacity (99%), ultrafine fibrous network and

high tensile strength (17, 22). Furthermore, it exhibits excellent biocompatibility and non-

toxicity, being a great material to be employed in a wide range applications in different areas

(6, 11, 69).

Bacterial cellulose/poly(methacrylate) nanocomposites have been receiving attention

by researchers due to their availability and attractive properties. These properties can be

employed in nanocomposites for biomedical applications such as wound dressing (43) and

design of 3D matrices (40). Glycidylmethacrylate (GMA) and

poly(ethyleneglycol)methacrylate (PEGMA) are two attractive methacrylate due to their

properties. GMA possess two functional groups, the epoxide and the methacrylate groups

(double bonds), which offers a wide range of industrial applications in polymer chemistry

and technology (49, 50, 54). PEGMA, one poly(ethyleneglycol) derivate, confers an

amphiphilic nature, which comes from its water-soluble PEG side chain with a pendant

hydroxyl group and its hydrophobic methacrylate group (62).

In this paper, the modification of the surface properties of bacterial cellulose with the

methacrylate compounds (glycidylmethacrylate and poly(ethyleneglycol)methacrylate)

through in situ radical polymerization was evaluated. Thermodynamic and morphologic

parameters such as dispersive and specific surface energy, acid–base surface character,

surface area and heterogeneity were determined using Inverse Gas Chromatography (IGC).

1.1. Inverse gas chromatography

Physical chemical properties of the surface nanocomposites can be determinated by

IGC. This technique is simple, fast and sensitive, giving great accuracy of the results. Unlike



72

GC, in IGC the nonvolatile material to be investigated is immobilized in a chromatographic

column (stationary phase) and through passage of molecules probe with known properties, it

is obtained the characteristic of the solid, at infinite dilution (Figure 42). Thermodynamic

parameters such as surface energy, acid-basic character, enthalpy, entropy, adsorption

isotherm, adsorption potential, heterogeneity and kinetic parameters such as diffusion,

permeability and solubility can be determined through this technique (87, 88).

Figure 42. Schematic representation of GC and IGC measurements (89).

1.1.1. Surface energy component

The surface energy of a solid, 𝛾𝑠, characterize the active energy in the surface of the

solid and can be given as the sum of the dispersive component, 𝛾𝑠𝑑, which describe all the van

der Waals forces interactions and specific component, 𝛾𝑠𝑠𝑝

, which express acid-base character

(87):

𝛾𝑠 = 𝛾𝑠𝑑 + 𝛾𝑠

𝑠𝑝 (Equation 6)



73

The retention time obtained from the interaction between the probe molecules and the

surface of the solid allows us to obtain the volume of probe molecules used, that is directly

related to the energy of adsorption (90), ∆𝐺𝑎𝑑𝑠:

∆𝐺𝑎𝑑𝑠 = 𝑅𝑇 ln 𝑉𝑁 + 𝐾 (Equation 7)

where 𝑅 is the gas constant, 𝐾 is a constant depending on the chosen reference state,

𝑇 is the column temperature and 𝑉𝑁 is the net retention volume. According Fowkes et al,

∆Gads is related to the energy of adhesion, 𝑊𝐴, by the equation:

𝑅𝑇𝑙𝑛𝑉𝑁 = 2𝑁𝐴(𝛾𝑠𝑑)1 2⁄ 𝑎(𝛾𝐿

𝑑)1 2⁄

+ 𝐾 (Equation 8)

where 𝛾𝐿𝑑 is the dispersive component of the surface energy of the probe molecule, 𝑎 is

the area occupied by probe molecule and 𝑁 is the Avogadro’s number. 𝛾𝑠𝑑 can be calculated

from the slope of the obtained line made a plot of ∆G𝑎𝑑𝑠 versus 𝑎(𝛾𝐿𝑑)

1 2⁄ of the series of n-

alkanes.

1.1.2. Acid base character

Knowledge of the surface materials acid or basic character is important to understand

their interaction with polar molecules. The polar molecules and solid interactions involve:

dispersive, ∆𝐺𝑎𝑑𝑠𝑑 , and specific, ∆𝐺𝑎𝑑𝑠

𝑠𝑝, interactions (92). The specific energy of adsorption

∆𝐺𝑎𝑑𝑠 is determined by the equation:

∆𝐺𝑎𝑑𝑠 = ∆𝐺𝑎𝑑𝑠𝑑 + ∆𝐺𝑎𝑑𝑠

𝑠𝑝 (Equation 9)

∆𝐺𝑎𝑑𝑠𝑠𝑝

can be determined by the following relation (92):

∆𝐺𝑎𝑑𝑠𝑠𝑝 = 𝑅𝑇𝑙𝑛𝑉𝑁 − 𝑅𝑇𝑙𝑛𝑉𝑁(𝑟𝑒𝑓) (Equation 10)



74

where 𝑉𝑁 is the net retention volume for the polar probe and VN(ref) is the net retention

volume established by the n-alkane reference line for the same polar probe. From ∆𝐺𝑎𝑑𝑠 it’s

possible to determine the surface enthalpy of adsorption, ∆𝐻𝑎𝑑𝑠 and surface entropy of

adsorption, ∆𝑆𝑎𝑑𝑠 through the following relation (92):

∆𝐺𝑎𝑑𝑠

𝑇=

∆𝐻𝑎𝑑𝑠

𝑇− ∆𝑆𝑎𝑑𝑠 (Equation 11)

∆𝐻𝑎𝑑𝑠𝑠𝑝

and ∆𝑆𝑎𝑑𝑠𝑠𝑝

can be established using the ∆Gadssp

in the previous equation. ∆Gadssp

can be used to quantify the Lewis acidity and basicity of the non-volatile material with the

following equation (92):

∆𝐻𝑎𝑑𝑠𝑠𝑝

𝐴𝑁∗=

𝐷𝑁

𝐴𝑁∗ × 𝐾𝐴 + 𝐾𝐵 (Equation 12)

where 𝐴𝑁∗ and 𝐷𝑁 are Gutmann’s (83) modified acceptor and donor numbers,

respectively; 𝐾𝐴 is a Lewis acidity constant and 𝐾𝐵 is a Lewis basicity constant.

1.1.3. Isotherm measurements

Using a wide variety of probe molecules at different temperatures, it’s obtained the

adsorption isotherm by the BET equation (93):

𝑝

𝑛(𝑝°−𝑝)=

1

𝑛𝑚𝑐+

𝑐−1

𝑛𝑚𝑐×

𝑝

𝑝° (Equation 13)

where nm is the monolayer capacity; 𝑛 the amount adsorbed; 𝑝 the partial pressure;

𝑝° the saturation pressure and 𝑐 is related with the heat of sorption. Knowing the monolayer

capacity and the cross area, 𝑎𝑚, of a probe molecule, the surface area, SBET, can be calculated

by the following equation (93):

𝑆𝐵𝐸𝑇 = 𝑎𝑚𝑛𝑚𝑁𝐴 (Equation 14)



75

where 𝑁𝐴 is the Avogadro constant.

Through the adsorption isotherm, the heterogeneity of the surface can be deduced

from the adsorption potential, 𝐴 by the following equation:

𝐴 = 𝑅𝑇𝑆𝑙𝑛 (𝑝°

𝑝) (Equation 15)

2. Experimental methodology

2.1. Material

Wet bacterial cellulose (BC) membranes in standard conditions were used (70).

Glycidyl methacrylate (GMA, 97%, with 100 ppm of monomethyl ether hydroquinone as

inhibitor), poly (ethylene glycol) methacrylate (PEGMA, average Mn 500, containing

900 ppm monomethyl ether hydroquinone as inhibitor), N,N’-methylenebisacrilamide

(MBA, ≥ 99.5%) and ammonium persulphate (APS, 98%) were purchase in Sigma-Aldrich

and were used as received. The chemical reagents used for IGC measurements such as non-

polar and polar molecules were GC grade (>99% purity) supplied by Sigma–Aldrich. The

methane gas (reference probe) and helium (carrier gas), of high purity (>99.99%), were

supplied by Air Liquide Company.

2.2. BC/PGMA and BC/PEGMA nanocomposites

The bacterial cellulose nanocomposites were obtained through the methodology

described in “Section 2.2. Chapter II: Bacterial cellulose/poly (glycidyl-methacrylate) nanocomposites

films by in situ radical polymerization” and Chapter IV: In situ radical polymerization of bacterial

cellulose/poly(ethyleneglycol)methacrylate nanocomposites films”

Briefly, wet membranes were drained to remove the excess of water. In a separate

Erlenmeyer, the membrane and the reaction mixture were purged in nitrogen for 30 min.

being added, posteriorly through a syringe. After 1 hour at room temperature, the reaction



76

took place in an oil bath for 6 hours at 60ºC. The BC nanocomposites prepared were washed

during 1 hour several times, dried at 40ºC and kept in a desiccator until their characterization.

The reaction mixture to the BC/PGMA was prepared in an aqueous solution with

(1:2) ratio of monomer, BC:GMA, (𝑤 𝑤⁄ ), initiator, APS, 0.5% (𝑤𝑖𝑛𝑖𝑡𝑖𝑎𝑡𝑜𝑟 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ ) and

cross-linking, MBA, 20% (𝑤𝑐𝑟𝑜𝑠𝑠𝑙𝑖𝑛𝑘𝑒𝑟 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ ) (when used).

For BC/PEGMA nanocomposites, the reaction mixture was prepared in an aqueous

solution with (1:20) ratio BC:PEGMA (𝑤 𝑤⁄ ) and initiator, 0.5% (𝑤𝑖𝑛𝑖𝑡𝑖𝑎𝑡𝑜𝑟 𝑤𝑚𝑜𝑛𝑜𝑚𝑒𝑟⁄ ).

2.3. IGC measurements

IGC measurements were carried out on a commercial inverse gas chromatograph

(iGC, Surface Measurements Systems, London, UK) equipped with flame ionization (FID)

and thermal conductivity (TCD) detectors. The iGC system is fully automatic with SMS iGC

Controller v1.8 control software. Data were analysed using iGC Standard v1.3 and

Advanced Analysis Software v1.25.

The sample were packed in the standard glass silanized (dymethyldichlorosilane;

Repelcote BDH, UK) columns with 0.2 cm ID and 30 cm in length by vertical tapping about

2 h. The columns with the samples were conditioned 8 h at 40 ºC and 2 h at the measurements

conditions, to stabilize the columns. After conditioning, pulse injections were carried out

with a 0.25 ml gas loop. Four n-alkanes (heptane, octane, nonane and decane) were used for

measurements of the dispersive component of surface energy at 20, 25 and 30 ºC. Four polar

probes (tetrahydrofuran, dichloromethane, acetonitrile and ethyl acetate) were used to

determine the specific component of surface energy and acid-base character (𝐾𝐴 and 𝐾𝐵) at

25 ºC. The physical constants for the probes used in IGC are presented in Table 7. The

isotherm experiments were undertaken with n-octane, tetrahydrofuran and dichloromethane

at 25 ºC. All experiments were carried out at 0% relative humidity with a helium flow rate of

10 ml/min at least in duplicate, producing a reproducibility error of less than 4%.



77

Table 7. Physical constants of all probes used in IGC experiments.

Probe 𝒂(10-19 m2) 𝜸𝑳𝒅(mJ/m2) 𝑫𝑵(kcal/mol) 𝑨𝑵∗(kcal/mol) 𝑫𝑵 𝑨𝑵∗⁄

n-Heptane 5.73 18.4 - - -

n-Octane 6.30 21.3 - - -

n-Nonane 6.90 22.7 - - -

n-Decane 7.50 23.4 - - -

Dichloromethane 2.45 24.5 0.0 20.4 -

Acetonitrile 2.14 27.5 14.1 4.7 3.00

Ethyl Acetate 3.30 19.6 17.1 1.5 11.4

Tetrahydrofuran 2.90 22.5 20.0 0.5 40.0

a: Cross-sectional area; γLd: surface tension; AN∗: electron acceptor number [0.288(AN − ANd)]; DN: electron donor number.

(83, 94)

2.4. Swelling ratio

The swelling ratio (𝑆𝑊) of the membranes was determined by immersing the samples

in distilled water at room temperature with a minimum of three replicates. The weight was

periodically measured during 48 hours. For each measurement the samples were taken out

of the water, their wet surfaces immediately wiped dry in filter paper, weigh, and then re-

immersed. The SW was calculated using the equation below:

𝑆𝑊(%) =(𝑊𝑠−𝑊𝑑)

𝑤𝑑 × 100% (Equation 16)

where, 𝑊𝑆 is the weight of bacterial cellulose after swelling and 𝑊𝑑 is the weight of

dry bacterial cellulose before swelling.

2.5. Scanning electron microscopy

Scanning electron microscopy (SEM) micrographs of the BC nanocomposite surfaces

were obtained on a HR-FESEM SU-70 Hitachi equipment operating at 1.5 kV in the field

emission mode. Samples were deposited on a steel plate and coated with carbon before

analysis.



78

2.6. Energy-dispersive X-ray spectroscopy

The semi-quantitative elemental chemical compositions of the samples were

determined by energy-dispersive X-ray spectroscopy (EDX). Semi-quantitative analyses (wt.

%) were done for carbon, oxygen and nitrogen and the 𝐶 𝑂⁄ ratio was obtained. EDX

experiments were conducted at an accelerated voltage of 5 kV in a Hitachi SU 8090

equipment.


IGC measurement allows to know the changes that occur in the materials surface

properties due to different modification processes. This data enables the knowledge of their

compatibility and behaviour with other molecules and modifications agents.

In order to study the influence of the poly(glycidylmethacrylate) (PGMA) and

poly(ethyleneglycol)methacrylate (PPEGMA), on the surface properties of the bacterial

cellulose (BC) produced by Gluconacetobacter sacchari, native BC and BC nanocomposites

were subjected to inverse gas chromatography (IGC) analysis. The dispersive component of

the surface energy, 𝛾𝑠𝑑, specific component of the surface energy, ∆𝐺𝑠

𝑠𝑝, surface area, 𝑆𝐵𝐸𝑇,

monolayer capacity, 𝑛𝑚, adsorption potential, 𝐴𝑚𝑎𝑥., acid character, 𝐾𝐴 and basic character,

𝐾𝐵 were determined.

3.1. Dispersive component of surface energy

𝛾𝑠𝑑 was determined by the injection of non-polar molecules (n-alkanes probes) and

obtained plotting 𝑅𝑇𝑙𝑛𝑉𝑛 against 𝑎(𝛾𝐿𝑑)

1/2 at different temperatures (Eq. 8). Table 8 shows

the results obtained for 𝛾𝑠𝑑 in native BC and BC methacrylate nanocomposites. The

correlation coefficient was good, ranging between 0.987 and 0.998.



79

Table 8. Dispersive component of surface energy, 𝛾𝑠𝑑, swelling ratio, 𝑆𝑊, of BC, BC/PGMA and BC/PPEGMA

nanocomposites.

Samples 𝜸𝑺

𝑫(mJ/m2) 𝑺𝑾 (%)

20 ºC 25 ºC 30 ºC

BC 58.53 51.8 47.41 137.84

BC/PGMA 59.10 55.21 51.96 32.80

BC/PGMA/MBA 88.25 82.93 78.31 31.36

BC/PPEGMA 35.54 35.42 36.24 431.64

The BC under study, produced by Gluconacetobacter sacchari, displays an higher 𝛾𝑠𝑑 than

those reported by Castro et al (39.64 mJ/m2) produced by Gluconacetobacter medellensis (38).

This can be attributed to the biosynthesis process of bacterial cellulose. The bacteria strain

and synthesis conditions can influence the cellulose chains arrangement and consequently

the crystallinity of BC. Cellulose type I is the common form produced by the bacteria

exhibiting two allomorph forms: monoclinic 𝐼𝛼 allomorph and triclinic 𝐼𝛽 form,

distinguishing themselves by their spatial arrangement of nanofibrils (9, 11, 13, 14). In this

particular case, the results suggest that the relative 𝐼𝛽 form is higher than 𝐼𝛼 allomorph (as

shown in Section 2.1. of Chapter II. New Bacterial cellulose/poly(glycidylmethacrylate)

nanocomposites films through in situ radical polymerization) which is more stable (38).

The incorporation of PGMA, an epoxide methacrylate monomer, leads to an increase

of 7% in the 𝛾𝑠𝑑 value. This result suggest that the in situ radical polymerization that occurs

inside of BC didn’t significantly affect the dispersive component of the surface. This inside

incorporation can be observed through the SEM images [Figure 43 (B)]. Furthermore, the

obtained swelling values for BC/PGMA/MBA reinforce the idea that the bacterial cellulose

increase their hydrophobicity probably due to the intra- and inter-chain hydrogen bonding

with PGMA which make this active sites less accessible to water absorption. Figure 44 shows

a schematic illustration of apolar and polar probe interactions with BC/PGMA/MBA

nanocomposites at surface.

With the addition of crosslinker, MBA, it was observed an increase of 51% in the 𝛾𝑠𝑑

value. Yang and Zhai (2012) reported that the water contact angle increased in cellulose fibers

grafted with GMA, due to the replacement of the polar hydroxyl groups by glycidyl groups,



80

ester and epoxy groups, that exhibit a less polar character. In the present study, the in situ free

radical polymerization of GMA crosslinked with MBA inside BC network probably leads to

two effects: (i) the increase in the number of glycidyl groups exposed in the surface which

present a less polar character, making it more hydrophobic or/and (ii) the change of the

spatial arrangement of microfibrils leaving them more exposed to surface (as shown in Figure

43 (C)).

Figure 43. SEM images of BC nanocomposites morphology (A) BC; (B) BC/PGMA; (C) BC/PGMA/MBA and (D) BC/PPEGMA.

However, the incorporation of the PPEGMA, change differently the surface

properties of BC. PPEGMA has an amphiphilic nature which arises from the pendant

hydroxyl group and its hydrophobic methacrylate group. BC/PPEGMA nanocomposite

exhibited a decrease in the 𝛾𝑠𝑑 value to 36.42 mJ/m2 at 25 ºC (40% less). This result suggests

that the incorporation of PPEGMA decreases the number of non-polar active sites probably

due to the increase of ester bonds and hydroxyls groups from PPEGMA exposed at

nanocomposite surface. As shown in SEM images [Figure 43 (D)], the BC/PPEGMA

exhibited a dense surface arising of incorporation which probably favours the probes

interaction with the polar active sites. The decrease in the 𝐶 𝑂⁄ ratio (2.90) in the



81

BC/PPEGMA nanocomposites compared to 𝐶 𝑂⁄ ratio (3.30) in the native BC evidence this

supposition. Besides that, the water retention ability shows an increase of 213 % compared

with native BC which means that the BC, acquire a hydrophilic character after incorporation

of PPEGMA, in agreement with the 𝛾𝑠𝑑 decrease.

Figure 44. Schematic interactions of non-polar and polar probes with surface groups of BC/PGMA/MBA nanocomposites.

3.2. Heterogeneity

The dispersive energetic profile of the surface heterogeneity is determined by BET

model (81) injecting different concentrations of a n-octane probe. The heterogeneity profile

for BC and BC nanocomposites at 25 ºC are shown in Figure 45. BC/PGMA/MBA

nanocomposites exhibited an increase of the actives sites energy, opposite to the

BC/PPEGMA nanocomposites where occurs a decrease of active sites energy when

compared with native BC. This behaviour is aligned with the results obtained to the

dispersive component of surface energy and described previously.



82

Figure 45. Heterogeneity profile with n-octane for native BC, (A) BC/PGMA and BC/PGMA/MBA and (B)

BC/PPEGMA nanocomposites at 25 ºC.

The BC/PGMA show one peak maximum (𝐴𝑚𝑎𝑥) at 12.59 kJ/Mol with a high

number of actives sites. In the presence of the crosslinker, BC/PGMA/MBA, present two

peak maximum, (𝐴𝑚𝑎𝑥) at 12.58 and 14.76 kJ/Mol, significantly more energetics compared

with native BC [Figure 45 (A)]. The addition of crosslinker plays an important function in

retaining more monomer into the BC network, having a great effect in 𝛾𝑠𝑑, making the surface

with more energetic dispersive actives sites which increase significantly the 𝛾𝑠𝑑. This non-

polar character probably comes from the alkanes chains introduced by more polymerization

of GMA being more affordable to the nanocomposite surface (as supported by SEM images

– Figure 43).



83

In the BC/PPEGMA nanocomposites one peak maximum (𝐴𝑚𝑎𝑥) at 7.38 kJ/Mol is

found with an increase number of less energetic actives sites [Figure 45 (B)], which make the

nanocomposite have smaller 𝛾𝑠𝑑. This result suggests that the introduction of PPEGMA into

the BC network leads to more accessible the non-polar PEG groups.

3.3. Surface area

The isotherm measurements with n-octane at different concentrations BET model

(81)were performed. Symmetrical peaks were obtained and a linear adsorption isotherm

described by Henry’s Law was observed. These indicates that the interactions between

materials and the probe molecule occurs predominantly via the high-energy sites (93).

Besides that a type I isotherm was observed, which is characteristic for microporous materials

with relatively small external surfaces (96).

The surface area (𝑆𝐵𝐸𝑇 ) of bacterial cellulose range between 0.83 m2/g and 12.59 m2/g

and the monolayer capacity (𝑛𝑚) range between 2.18 µmol/g and 33.17 µmol/g (Table 9).

These results show that the polymerization with the two methacrylates had different effects

in the particle morphology. BC/PGMA and BC/PPEGMA nanocomposites increased the

𝑆𝐵𝐸𝑇 in 57 % and 219 %, respectively, when compared with native BC, which means: (i) a

significant decrease in the particles size or/and (ii) increase in its porosity or roughness (89).

SEM images (Figure 43) show that the porosity increase when both polymers were added to

BC network.

The addition of crosslinker had an opposite behaviour, decreasing significantly the

𝑆𝐵𝐸𝑇 in 79 %, which means that occurs an increase of particle size or/and a decrease in

porosity/roughness. This can be due to the more compact and dense surface formed after

incorporation of PGMA/MBA as supported by SEM images (Figure 43).



84

Table 9. Surface area, 𝑆𝐵𝐸𝑇, monolayer capacity, 𝑛𝑚 and adsorption potential distribution maximum, 𝐴𝑚𝑎𝑥, of BC,

BC/PGMA, PC/PGMA/MBA and BC/PPEGMA nanocomposites, at 25 ºC with n-octane.

Samples 𝑺𝑩𝑬𝑻 (m2/g) 𝒏𝒎 (µMol/g) R2

BC 3.94 10.38 0.9994

BC/PGMA 6.20 16.35 0.9989

BC/PGMA/MBA 0.83 2.18 0.9957

BC/PPEGMA 12.59 33.17 0.9499

3.4. Acid-base character

BC, GMA and PEGMA have in its structure polar groups able to interact and

exchange the specific interactions with polar probes. Injecting tetrahydrofuran,

dichloromethane, ethyl acetate and acetonitrile, the specific component of surface energy,

∆𝐺𝑠𝑠𝑝

, was determined and is showed in Figures 46 and 47, respectively.

The high interaction between acetonitrile (amphoteric probes) and the

nanocomposites evidence the presence of both acid and basic groups in the nanocomposites

surface.

To the BC/PGMA nanocomposites the ∆𝐺𝑠𝑠𝑝

increase for all probes which suggest

that occurs an increase in polar active sites at the surface. This increase is more significant

for the acid probe (dichloromethane) compared to the basic probe (tetrahydrofuran),

indicating of an increase in the basic active sites in the surface. The energetic profile of

dichloromethane showed that it occurs due to an increase of the active sites number with

high energy [Figure 48 (A)]. These results suggest that the incorporation of monomer and the

addition of crosslinker affects the acid-base surface character making more basic groups at

the surface.



85

Figure 46. Specific surface energy of BC, BC/PGMA and BC/PGMA/MBA nanocomposites with polar probes: THF -

tetrahydrofuran, DCM - dichloromethane, ETOAc – ethyl acetate and ACN – acetonitrile, at 25 ºC.

Figure 47. Specific surface energy of BC and BC/PPEGMA nanocomposites with polar probes: THF - tetrahydrofuran,

DCM - dichloromethane, ETOAc – ethyl acetate, ACN – acetonitrile, at 25 ºC.



86

Figure 48. Heterogeneity profile with dichloromethane for (A) BC, BC/PGMA and BC/PGMA/MBA and (B) BC and BC/PPEGMA nanocomposites, at 25 ºC.

To the BC/PPEGMA nanocomposites, a higher increase in the ∆𝐺𝑠𝑠𝑝

for

dichloromethane (Figure 47) was observed. The dichloromethane energetic profile [Figure

48 (B)] show an increase in the number of active sites. This observation suggests more basic

groups at nanocomposites surface accessible for interactions.

The calculated ∆𝐺𝑠𝑠𝑝

is converted into acid-base number using the Guttmann’s

concept (83). Table 10 present the obtained values for acid-base constants, 𝐾𝐴 and 𝐾𝐵, for

bacterial cellulose nanocomposites. The surface basicity exhibited in native BC is higher than

the one found by Castro et al (2015), which reported a 𝐾𝐵 𝐾𝐴⁄ ratio of 1.80. This result suggests



87

a different arrangement of the cellulose groups at the surface with less hydroxyls groups

available to interactions. The incorporation of basic groups such as (C–O–C) in BC/PGMA

and BC/PPEGMA nanocomposites leads to an increase of the base constant, 𝐾𝐵, showing a

𝐾𝐵 𝐾𝐴⁄ ratio about four times higher.

Taking into account the energetic profile, the increase in the basic character

exhibited in BC nanocomposites derives from the increased number of more energetic basic

active sites due to the incorporation of methacrylates compounds into BC network.

Table 10. Acid-base character, 𝐾𝐴 and 𝐾𝐵 of BC, BC/PGMA BC/PGMA/MBA and BC/PPEGMA nanocomposites, at

25 ºC.

Samples 𝑲𝑨 𝑲𝑩 𝑲𝑩

𝑲𝑨⁄ R2

BC 0.10 0.23 2.27 0.9873

BC/PGMA 0.10 0.42 4.20 0.9868

BC/PGMA/MBA 0.13 0.56 4.31 0.9862

BC/PPEGMA 0.08 0.34 4.25 0.9668

4. Conclusions

The incorporation of methacrylate monomers into the BC network through in situ

radical polymerization is a suitable method to change the BC properties for the production

of new nanocomposites aiming new applications. Inverse Gas Chromatography shows that

the incorporation of glycidylmethacrylate and poly(ethyleneglycol)methacrylate modify the

bacterial cellulose which leads:

(i) in BC/poly(glycidylmethacrylate), to an increase in the dispersive component

of surface energy (decrease in hydrophilicity) and an decrease the surface area

when the crosslinked was added;

(ii) in BC/(poly(ethylene glycol)methacrylate), to a significant decrease in the in the

dispersive component of surface energy (increase of hydrophilicity) and a

decrease in surface area;



88

(iii) in both BC/methacrylate nanocomposites, to a great increase of the basic

surface character.

89

FINAL CONCLUSIONS

BC as support for nanocomposite films has been exploited for numerous applications

and their combination with methacrylate monomers allow to obtain new nanomaterials with

different properties to apply in various fields, namely biomedical applications or/and remove

heavy metals.

In situ free radical polymerization of glycidylmethacrylate, GMA, and poly (ethylene

glycol) methacrylate, PEGMA, occurred into the BC network using ammonium persulphate,

APS, as initiator and N,N’-methylenebisacrilamide, MBA, as crosslinker in BC/PGMA. The

optimal conditions determined for polymerization was (1:2) ratio for BC/PGMA, (1:2:0.2)

for BC/PGMA/MBA and (1:20) for BC/PPEGMA with 0.5 % of initiator during 6 h at 60

ºC. A maximum incorporation percentage of 60, 67 and 87 %, were obtained respectively.

Compared with native BC, the BC/PGMA nanocomposites exhibited a roughness and a

compact three-dimensional structure, an improvement in thermal and mechanical properties,

a decrease in their swelling ability and a lower crystallinity. Unlike, the BC/PPEGMA had

shown a decrease of stiffness of three-dimensional structure and an increase in their swelling

ability. However, an improvement in thermal and mechanical properties and an increase in

the amorphous content it’s observed. Both BC nanocomposites present basic surface

character.

Chemical treatment proved to be a good and simple strategy to modified BC/PGMA

nanocomposites through epoxide ring-opening reaction mechanism. The replacement of

hydroxyls groups of cellulose chains occurs for the linkage of GMA chains by epoxy group.

Nanocomposites became more compact, smooth and with more water retention ability. A

decrease in the thermal and mechanical proprieties was observed.

As future perspectives, the reactivity of epoxy group as absorbent material, it’s an

interesting as well as the biocompatibility of all BC polymethacrylate nanocomposites for

biomedical applications for application as adsorbent material for removal of heavy metal

or/and proteins.

90

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